Methods and compositions related to combined treatment with anti-inflammatories and synthetic nanocarriers comprising an immunosuppressant

ABSTRACT

Provided herein are methods and compositions, such as kits, related to compositions comprising synthetic nanocarriers comprising an immunosuppressant and compositions comprising an uricase and a composition comprising an anti-inflammatory therapeutic. Also provided herein are methods and compositions for the treatment of subjects in need of administration or treatment with the uricase.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119 ofU.S. Provisional Application 62/470,250 filed Mar. 11, 2017, the entirecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

Provided herein are methods and compositions, such as kits, related tocompositions comprising synthetic nanocarriers comprising animmunosuppressant and compositions comprising an uricase and acomposition comprising an anti-inflammatory therapeutic. Also providedherein are methods and compositions for the treatment of subjects inneed of administration or treatment with the uricase.

SUMMARY OF THE INVENTION

In one aspect, a method, comprising concomitantly administering to asubject in need thereof 1) a composition comprising syntheticnanocarriers comprising an immunosuppressant and 2) a compositioncomprising an uricase; and further comprising administering 3) acomposition comprising an anti-inflammatory therapeutic, wherein thecomposition comprising an anti-inflammatory therapeutic is administeredconcomitantly with the composition comprising synthetic nanocarrierscomprising an immunosuppressant and the composition comprising anuricase is provided.

In one embodiment of any one of the methods or compositions providedherein the composition comprising an anti-inflammatory therapeutic isadministered prior to the composition comprising synthetic nanocarrierscomprising an immunosuppressant and the composition comprising anuricase. In one embodiment of any one of the methods or compositionsprovided herein the anti-inflammatory therapeutic is administered atleast once prior. In one embodiment of any one of the methods orcompositions provided herein the anti-inflammatory therapeutic isadministered a week prior.

In one embodiment of any one of the methods or compositions providedherein the anti-inflammatory therapeutic is a NSAID. In one embodimentof any one of the methods or compositions provided herein theanti-inflammatory therapeutic is colchicine or ibuprofen.

In one embodiment of any one of the methods provided herein the methodfurther comprises administering to the subject one or more compositionscomprising an infusion reaction therapeutic. In one embodiment of anyone of the methods provided herein the method comprises theadministration of at least two infusion reaction therapeutics.

In one embodiment of any one of the methods or compositions providedherein the infusion reaction therapeutic(s) comprise an antihistamineand/or a corticosteroid. In one embodiment of any one of the methods orcompositions provided herein the antihistamine is fexofenadine. In oneembodiment of any one of the methods or compositions provided herein thecorticosteroid is methylprednisolone, prednisone or dexamethasone.

In one embodiment of any one of the methods provided herein thecomposition(s) comprising an infusion reaction therapeutic is/areadministered concomitantly with the composition comprising syntheticnanocarriers comprising an immunosuppressant and the compositioncomprising an uricase. In one embodiment of any one of the methodsprovided herein the composition(s) comprising an infusion reactiontherapeutic is/are administered at least once prior to the compositioncomprising synthetic nanocarriers comprising an immunosuppressant andthe composition comprising an uricase. In one embodiment of any one ofthe methods provided herein the composition(s) comprising an infusionreaction therapeutic is/are administered at least twice prior to thecomposition comprising synthetic nanocarriers comprising animmunosuppressant and the composition comprising an uricase. In oneembodiment of any one of the methods provided herein the composition(s)comprising an infusion reaction therapeutic is/are administered within24 hours of the composition comprising synthetic nanocarriers comprisingan immunosuppressant and the composition comprising an uricase.

In one embodiment of any one of the methods or compositions providedherein the uricase is pegylated uricase. In one embodiment of any one ofthe methods or compositions provided herein the pegylated uricase ispegsiticase (i.e., pegadricase) or pegloticase. In one embodiment of anyone of the methods or compositions provided herein the immunosuppressantis an mTOR inhibitor. In one embodiment of any one of the methods orcompositions provided herein the mTOR inhibitor is a rapalog. In oneembodiment of any one of the methods or compositions provided herein therapalog is rapamycin.

In one embodiment of any one of the methods provided herein the subjectis human. In one embodiment of any one of the methods provided hereinthe subject is a subject with an elevated serum uric acid level and/orundesired uric acid deposits. In one embodiment of any one of themethods provided herein the subject has hyperuricemia. In one embodimentof any one of the methods provided herein the subject has gout or acondition associated with gout. In one embodiment of any one of themethods provided herein the subject has acute gout; chronic gout with orwithout tophi; idiopathic gout; refractory gout, such as chronicrefractory gout; secondary gout; unspecified gout; gout associated witha cardiovascular condition, renal condition, pulmonary condition,neurological condition, ocular condition, dermatological condition orhepatic condition; or has had a gout attack or gout flare.

In one embodiment of any one of the methods or compositions providedherein the immunosuppressant is encapsulated in the syntheticnanocarriers. In one embodiment of any one of the methods orcompositions provided herein the synthetic nanocarriers are polymericsynthetic nanocarriers. In one embodiment of any one of the methods orcompositions provided herein the polymeric synthetic nanocarrierscomprise a hydrophobic polyester. In one embodiment of any one of themethods or compositions provided herein the hydrophobic polyestercomprises PLA, PLG, PLGA or polycaprolactone. In one embodiment of anyone of the methods or compositions provided herein the polymericsynthetic nanocarriers further comprise PEG. In one embodiment of anyone of the methods or compositions provided herein the PEG is conjugatedto the PLA, PLG, PLGA or polycaprolactone. In one embodiment of any oneof the methods or compositions provided herein the polymeric syntheticnanocarriers comprise PLA, PLG, PLGA or polycaprolactone and PEGconjugated to PLA, PLG, PLGA or polycaprolactone. In one embodiment ofany one of the methods or compositions provided herein the polymericsynthetic nanocarriers comprise PLA and PLA-PEG.

In one embodiment of any one of the methods or compositions providedherein the mean of a particle size distribution obtained using dynamiclight scattering of the synthetic nanocarriers is a diameter greaterthan 120 nm. In one embodiment of any one of the methods or compositionsprovided herein the diameter is greater than 150 nm. In one embodimentof any one of the methods or compositions provided herein the diameteris greater than 200 nm. In one embodiment of any one of the methods orcompositions provided herein the diameter is greater than 250 nm. In oneembodiment of any one of the methods or compositions provided herein thediameter is less than 500 nm. In one embodiment of any one of themethods or compositions provided herein the diameter is less than 450nm. In one embodiment of any one of the methods or compositions providedherein the diameter is less than 400 nm. In one embodiment of any one ofthe methods or compositions provided herein the diameter is less than350 nm. In one embodiment of any one of the methods or compositionsprovided herein the diameter is less than 300 nm. In one embodiment ofany one of the methods or compositions provided herein the diameter isless than 250 nm. In one embodiment of any one of the methods orcompositions provided herein the diameter is less than 200 nm.

In one embodiment of any one of the methods or compositions providedherein the load of the immunosuppressant of the synthetic nanocarriersis 7-12% or 8-12% by weight. In one embodiment of any one of the methodsor compositions provided herein the load of the immunosuppressant of thesynthetic nanocarriers is 7-10% or 8-10% by weight. In one embodiment ofany one of the methods or compositions provided herein the load of theimmunosuppressant of the synthetic nanocarriers is 7%, 8%, 9%, 10%, 11%,or 12% by weight.

In one embodiment of any one of the methods provided herein theadministration of any one of or the set of compositions is repeated. Inone embodiment of any one of the methods provided herein the repeatedadministrations of the composition(s) are repeated on a monthly basis.In one embodiment of any one of the methods provided herein thecomposition comprising the anti-inflammatory therapeutic of eachrepeated administration is given as in any one of the methods providedherein relative to the time of the corresponding repeated administrationof the composition comprising synthetic nanocarriers comprising animmunosuppressant and the composition comprising an uricase. In oneembodiment of any one of the methods provided herein the composition(s)comprising the infusion reaction therapeutic(s) of each repeatedadministration is given as in any one of the methods provided hereinrelative to the time of the corresponding repeated administration of thecomposition comprising synthetic nanocarriers comprising animmunosuppressant and the composition comprising an uricase.

In one embodiment of any one of the methods provided herein theadministration of the composition comprising synthetic nanocarrierscomprising an immunosuppressant and the composition comprising anuricase is according to the doses and/or frequencies and/or timing,respectively, of any one of the methods provided herein. In oneembodiment of any one of the methods provided herein the administrationof the composition comprising synthetic nanocarriers comprising animmunosuppressant, the composition comprising an uricase, and thecomposition comprising an anti-inflammatory therapeutic is according tothe doses and/or frequencies and/or timing, respectively, of any one ofthe methods provided herein. In one embodiment of any one of the methodsprovided herein the administration of the composition comprisingsynthetic nanocarriers comprising an immunosuppressant, the compositioncomprising an uricase, the composition comprising an anti-inflammatorytherapeutic, and the one or more compositions comprising an infusionreaction therapeutic is according to the doses and/or frequencies and/ortiming, respectively, of any one of the methods provided herein. Any oneof the respective doses and/or frequencies and/or timing can be used forany one of the methods provided herein for the respective composition.

In one embodiment of any one of the methods provided herein theadministration of the composition comprising synthetic nanocarrierscomprising an immunosuppressant and the composition comprising anuricase is according to any one of the regimens, respectively, providedherein. In one embodiment of any one of the methods provided herein theadministration of the composition comprising synthetic nanocarrierscomprising an immunosuppressant, the composition comprising an uricaseand the composition comprising an anti-inflammatory therapeutic isaccording to any one of the regimens, respectively, provided herein. Inone embodiment of any one of the methods provided herein theadministration of the composition comprising synthetic nanocarrierscomprising an immunosuppressant, the composition comprising an uricase,the composition comprising an anti-inflammatory therapeutic and the oneor more compositions comprising an infusion reaction therapeutic isaccording to any one of the regimens, respectively, provided herein. Anyone of the respective regimens can be used for any one of the methodsprovided herein for the respective composition.

In one embodiment of any one of the methods provided herein theadministration of the composition comprising synthetic nanocarrierscomprising an immunosuppressant is given according the any one of themodes of administration provided herein. In one embodiment of any one ofthe methods provided herein the administration of the compositioncomprising an uricase is given according the any one of the modes ofadministration provided herein. In one embodiment of any one of themethods provided herein the administration of the composition comprisingan anti-inflammatory therapeutic is given according the any one of themodes of administration provided herein. In one embodiment of any one ofthe methods provided herein the administration of the composition(s)comprising an infusion reaction therapeutic is/are given according theany one of the modes of administration provided herein.

In one aspect a composition, comprising 1) a composition comprisingsynthetic nanocarriers comprising an immunosuppressant and 2) acomposition comprising an uricase; and further comprising administering3) a composition comprising an anti-inflammatory therapeutic isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image showing tophi/uric acid deposits visualized usingDECT.

FIG. 2 is a cartoon representation of the components of SEL-212.

FIG. 3 is a graph of mean serum uric acid (sUA) levels in the 5 cohortsof the phase 1a clinical trial following a single intravenous infusionof pegsiticase.

FIG. 4 is a graphical illustration showing the serum uric acid levelsand uricase-specific ADA levels for each subject in Cohort #3 of thePhase 1a clinical trial and Cohort #9, Cohort # 4, and Cohort #6 in thePhase 1b clinical trial.

FIG. 5 is a graph showing the serum uric acid levels of Cohort #3 fromthe Phase 1a clinical and Cohort #9, Cohort #1, Cohort #2, Cohort #3,Cohort #4, Cohort #5 and Cohort #6 from the Phase 1b clinical trialtrial.

FIG. 6 from left to right shows data from two replicate Kystexxa®trials, in the middle is the data of SVP-Rapamycin alone vs. pegsiticasealone (Cohort #9) and then Rapamycin alone vs. Cohort #6 (a SEL-212cohort).

FIG. 7 is a graphical illustration showing the serum uric acid levels ofsubjects treated with pegstiticase alone, or in combination withsynthetic nanocarriers comprising rapamycin (SVP-Rapamycin) (0.1 or 0.3mg/kg).

FIG. 8 shows doses for the phase 2 clinical trial.

DETAILED DESCRIPTION OF THE INVENTION A. Overview

Anti-drug antibodies (ADAs) can be a complication with treatment withsoluble therapeutic proteins, such as the possibility of reducedefficacy. In addition, with treatment of subjects with conditionsassociate with gout, gout flares can increase when efficacy isincreased. It has been surprisingly found that the administration of ananti-inflammatory therapeutic with a composition comprising syntheticnanocarriers comprising an immunosuppressant and a compositioncomprising an uricase can result in better efficacy of treatment as wellas gout flare reduction. This unexpected outcome is significantly betterthan with other therapies as shown in Table 4. Specifically, acombination assessment of glare frequency as well as the area under thecurve of mean sUA (serum uric acid) levels over time was performed. Thefinding of both improved efficacy and reduced gout flares was surprisingwith the SEL-212 study drug and anti-inflammatory treatment.

B. Definitions

“Administering” or “administration” or “administer” means giving amaterial to a subject in a manner such that there is a pharmacologicalresult in the subject. This may be direct or indirect administration,such as by inducing or directing another subject, including anotherclinician or the subject itself, to perform the administration.

“Amount effective” in the context of a composition or dose foradministration to a subject refers to an amount of the composition ordose that produces one or more desired responses in the subject. In someembodiments, the amount effective is a pharmacodynamically effectiveamount. Therefore, in some embodiments, an amount effective is anyamount of a composition or dose provided herein that produces one ormore of the desired therapeutic effects and/or immune responses asprovided herein. This amount can be for in vitro or in vivo purposes.For in vivo purposes, the amount can be one that a clinician wouldbelieve may have a clinical benefit for a subject in need thereof. Anyone of the compositions or doses, including label doses, as providedherein can be in an amount effective.

Amounts effective can involve reducing the level of an undesiredresponse, although in some embodiments, it involves preventing anundesired response altogether. Amounts effective can also involvedelaying the occurrence of an undesired response. An amount that iseffective can also be an amount that produces a desired therapeuticendpoint or a desired therapeutic result. In other embodiments, theamounts effective can involve enhancing the level of a desired response,such as a therapeutic endpoint or result. Amounts effective, preferably,result in a therapeutic result or endpoint and/or reduced or eliminatedADAs against the treatment and/or reduced gout flare frequency incombination with improved efficacy in any one of the subjects providedherein. The achievement of any of the foregoing can be monitored byroutine methods.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason.

Doses of the components in any one of the compositions of the inventionor used in any one of the methods of the invention may refer to theamount of the components in the composition, the actual amounts of therespective components received by an administered subject, or the amountthat appears on a label (also referred to herein as label dose). Thedose can be administered based on the number of synthetic nanocarriersthat provide the desired amount of the component(s).

“Antihistamine” refer to agents that block the effects of histamine.

“Attach” or “Attached” or “Couple” or “Coupled” (and the like) means tochemically associate one entity (for example a moiety) with another. Insome embodiments, the attaching is covalent, meaning that the attachmentoccurs in the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent attaching ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of attaching.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“Concomitantly” means administering two or more materials/agents to asubject in a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in a physiologic orimmunologic response, and even more preferably the two or morematerials/agents are administered in combination. In embodiments,concomitant administration may encompass administration of two or morematerials/agents within a specified period of time, preferably within 1month , more preferably within 1 week, still more preferably within 1day, and even more preferably within 1 hour. In embodiments, the two ormore materials/agents are sequentially administered. In embodiments, thematerials/agents may be repeatedly administered concomitantly; that isconcomitant administration on more than one occasion.

“Dose” refers to a specific quantity of a pharmacologically activematerial for administration to a subject for a given time. Unlessotherwise specified, the doses recited for compositions comprisingpegylated uricase refer to the weight of the uricase (i.e., the proteinwithout the weight of the PEG or any other components of the compositioncomprising the pegylated uricase). Also, unless otherwise specified, thedoses recited for compositions comprising synthetic nanocarrierscomprising an immunosuppressant refer to the weight of theimmunosuppressant (i.e., without the weight of the synthetic nanocarriermaterial or any of the other components of the synthetic nanocarriercomposition). When referring to a dose for administration, in anembodiment of any one of the methods, compositions or kits providedherein, any one of the doses provided herein is the dose as it appearson a label/label dose.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier. In embodiments of any one of themethods or compositions provided herein, the immunosuppressants areencapsulated within the synthetic nanocarriers.

“Elevated serum uric acid level” refers to any level of uric acid in asubject’s serum that may lead to an undesirable result or would bedeemed by a clinician to be elevated. In an embodiment, the subject ofany one of the methods provided herein can have a serum uric acid levelof ≥ 5 mg/dL, ≥ 6 mg/dL, or ≥ 7 mg/dL. Such a subject may be ahyperuremic subject. Whether or not a subject has elevated blood uricacid levels can be determined by a clinician, and in some embodiments,the subject is one in which a clinician has identified or would identifyas having elevated serum uric acid levels.

“Gout” generally refers to a disorder or condition associated with thebuildup of uric acid, such as deposition of uric crystals in tissues andjoints, and/or a clinically relevant elevated serum uric acid level.Accumulation of uric acid may be due to overproduction of uric acid orreduced excretion of uric acid. Gout may range from asymptomatic tosevere and painful inflammatory conditions. A “condition associated withgout” refers to any condition in a subject where the subject experienceslocal and/or systemic effects of gout, including inflammation and immuneresponses, and in which the condition is caused or exacerbated by, orthe condition can result in or exacerbate, gout. A gout flare is an“attack” or exacerbation of gout symptoms, which can happen at any time.Gout flares can include gout flares that occur after the administrationof a uric acid lowering therapy.

“Hydrophobic polyester” refers to any polymer that comprises one or morepolyester polymers or units thereof and that has hydrophobiccharacteristics. Polyester polymers include, but are not limited to,PLA, PLGA, PLG and polycaprolactone. “Hydrophobic” refers to a materialthat does not substantially participate in hydrogen bonding to water.Such materials are generally non-polar, primarily non-polar, or neutralin charge. Synthetic nanocarriers may be completely comprised ofhydrophobic polyesters or units thereof. In some embodiments, however,the synthetic nanocarriers comprise hydrophobic polyesters or unitsthereof in combination with other polymers or units thereof. These otherpolymers or units thereof may by hydrophobic but are not necessarily so.In some preferred embodiments, when synthetic nanocarriers include oneor more other polymers or units thereof in addition to a hydrophobicpolyester, the matrix of other polymers or units thereof with thehydrophobic polyester is hydrophobic overall. Examples of syntheticnanocarriers that can be used in the invention and that comprisehydrophobic polyesters can be found in U.S. Publication Nos. US2016/0128986 and US 2016/0128987, and such synthetic nanocarriers andthe disclosure of such synthetic nanocarriers is incorporated herein byreference.

“Immunosuppressant”, as used herein, means a compound that can cause atolerogenic immune response specific to an antigen, also referred toherein as an “immunosuppressive effect”. An immunosuppressive effectgenerally refers to the production or expression of cytokines or otherfactors by an antigen-presenting cell (APC) that reduces, inhibits orprevents an undesired immune response or that promotes a desired immuneresponse, such as a regulatory immune response, against a specificantigen. When the APC acquires an immunosuppressive function (under theimmunosuppressive effect) on immune cells that recognize an antigenpresented by this APC, the immunosuppressive effect is said to bespecific to the presented antigen. Examples of immunosuppressantsinclude “mTOR inhibitors”, a class of drugs that inhibit mTOR, aserine/threonine-specific protein kinase that belongs to the family ofphosphatidylinositol-3 kinase (PI3K) related kinases (PIKKs). mTORinhibitors include, but are not limited to, rapalogs, such as rapamycin,as well as ATP-competitive mTOR kinase inhibitors, such as mTORCl/mTORC2dual inhibitors.

In embodiments of any one of the methods, compositions or kits providedherein, the immunosuppressants provided herein are attached to syntheticnanocarriers. In preferable embodiments, the immunosuppressant is anelement that is in addition to the material that makes up the structureof the synthetic nanocarrier. For example, in one embodiment, where thesynthetic nanocarrier is made up of one or more polymers, theimmunosuppressant is a compound that is in addition and attached to theone or more polymers. In embodiments, such as where the material of thesynthetic nanocarrier also results in an immunosuppressive effect, theimmunosuppressant is an element present in addition to the material ofthe synthetic nanocarrier that results in an immunosuppressive effect.

“Infusion reaction therapeutics” are therapeutics that can be beneficialin reducing or preventing infusion reactions. Examples of suchtherapeutics include anti-inflammatories, such as corticosteroids.

“Load”, when comprised in a composition comprising a syntheticnanocarrier, such as coupled thereto, is the amount of theimmunosuppressant in the composition based on the total dry recipeweight of materials in an entire synthetic nanocarrier (weight/weight).Generally, such a load is calculated as an average across a populationof synthetic nanocarriers. In one embodiment, the load on average acrossthe synthetic nanocarriers is between 0.1% and 15%. In anotherembodiment, the load is between 0.1% and 10%. In a further embodiment,the load is between 1% and 15%. In yet a further embodiment, the load isbetween 5% and 15%. In still a further embodiment, the load is between7% and 12%. In still a further embodiment, the load is between 8% and12%. In still another embodiment, the load is between 7% and 10%. Instill another embodiment, the load is between 8% and 10%. In yet afurther embodiment, the load is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, or 15% on average across the population of synthetic nanocarriers.In any one of the methods, compositions or kits provided herein, theload of the immunosuppressant, such as rapamycin, may be any one of theloads provided herein.

The rapamycin (or other immunosuppressant) load of the nanocarrier insuspension can be calculated by dividing the rapamycin content of thenanocarrier as determined by HPLC analysis of the test article by thenanocarrier mass. The total polymer content is measured either bygravimetric yield of the dry nanocarrier mass or by the determination ofthe nanocarrier solution total organic content following pharmacopeiamethods and corrected for PVA content.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 µm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofsynthetic nanocarriers may vary depending on the embodiment. Forinstance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1.

Preferably, a maximum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample is equal to or less than 3 µm, more preferably equal to or lessthan 2 µm, more preferably equal to or less than 1 µm, more preferablyequal to or less than 800 nm, more preferably equal to or less than 600nm, and more preferably still equal to or less than 500 nm. In preferredembodiments, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm, more preferably equal to orgreater than 120 nm, more preferably equal to or greater than 130 nm,more preferably equal to or greater than 140 nm, and more preferablystill equal to or greater than 150 nm. Measurement of syntheticnanocarrier dimensions (e.g., effective diameter) may be obtained, insome embodiments, by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (DLS) (e.g.,using a Brookhaven ZetaPALS instrument). For example, a suspension ofsynthetic nanocarriers can be diluted from an aqueous buffer intopurified water to achieve a final synthetic nanocarrier suspensionconcentration of approximately 0.01 to 0.5 mg/mL. The diluted suspensionmay be prepared directly inside, or transferred to, a suitable cuvettefor DLS analysis. The cuvette may then be placed in the DLS, allowed toequilibrate to the controlled temperature, and then scanned forsufficient time to acquire a stable and reproducible distribution basedon appropriate inputs for viscosity of the medium and refractiveindicies of the sample. The effective diameter, or mean of thedistribution, is then reported. Determining the effective sizes of highaspect ratio, or non-spheroidal, synthetic nanocarriers may requireaugmentative techniques, such as electron microscopy, to obtain moreaccurate measurements. “Dimension” or “size” or “diameter” of syntheticnanocarriers means the mean of a particle size distribution, forexample, obtained using dynamic light scattering.

“Pegylated uricase” refers to any uricase that is attached to one ormore PEG (poly(ethylene glycol), poly (ethylene oxide) or poly(oxyethylene)) molecules (i.e., a poly(ethylene glycol), poly (ethyleneoxide) or poly (oxyethylene) polymer or unit thereof). Preferably insome embodiments, the one or more PEG molecules are poly(ethyleneglycol) molecules. The terms “pegylated” or “pegylation” refer to theconjugated form or the act of conjugating to the uricase, respectively.Such a modified uricase is referred to as pegylated uricase. Pegylateduricases include, but are not limited to pegsiticase (i.e., pegadricase)and pegloticase (KRYSTEXXA®).

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” means a pharmacologically inactive material used together witha pharmacologically active material to formulate the compositions.Pharmaceutically acceptable excipients comprise a variety of materialsknown in the art, including but not limited to saccharides (such asglucose, lactose, and the like), preservatives such as antimicrobialagents, reconstitution aids, colorants, saline (such as phosphatebuffered saline), and buffers. Any one of the compositions providedherein may include a pharmaceutically acceptable excipient or carrier.

“Rapalog” refers to rapamycin and molecules that are structurallyrelated to (an analog) of rapamycin (sirolimus), and are preferablyhydrophobic. Examples of rapalogs include, without limitation,temsirolimus (CCI-779), deforolimus, everolimus (RAD001), ridaforolimus(AP-23573), zotarolimus (ABT-578). Additional examples of rapalogs maybe found, for example, in WO Publication WO 1998/002441 and U.S. Pat.No. 8,455,510, the disclosure of such rapalogs are incorporated hereinby reference in its entirety. In any one of the methods or compositionsor kits provided herein, the immunosuppressant may be a rapalog.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like. In any one of the methods, compositions and kits providedherein, the subject is human. In any one of the methods, compositionsand kits provided herein, the subject is any one of the subjectsprovided herein, such as one that in is need thereof, such as in need oftreatment with an uricase.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Synthetic nanocarriers may be a variety ofdifferent shapes, including but not limited to spheroidal, cuboidal,pyramidal, oblong, cylindrical, toroidal, and the like. Syntheticnanocarriers comprise one or more surfaces.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Examples of syntheticnanocarriers include (1) the biodegradable nanoparticles disclosed inU.S. Pat. 5,543,158 to Gref et al., (2) the polymeric nanoparticles ofPublished U.S. Pat. Application 20060002852 to Saltzman et al., (3) thelithographically constructed nanoparticles of Published U.S. Pat.Application 20090028910 to DeSimone et al., (4) the disclosure ofWO2009/051837 to von Andrian et al., (5) the nanoparticles disclosed inPublished U.S. Pat. Application 2008/0145441 to Penades et al., (6) thenanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), and (7) those of Look et al., Nanogel-based delivery ofmycophenolic acid ameliorates systemic lupus erythematosus in mice” J.Clinical Investigation 123(4):1741-1749(2013).

Synthetic nanocarriers may have a minimum dimension of equal to or lessthan about 100 nm, preferably equal to or less than 100 nm, do notcomprise a surface with hydroxyl groups that activate complement oralternatively comprise a surface that consists essentially of moietiesthat are not hydroxyl groups that activate complement. In an embodiment,synthetic nanocarriers that have a minimum dimension of equal to or lessthan about 100 nm, preferably equal to or less than 100 nm, do notcomprise a surface that substantially activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not substantially activate complement. In a more preferredembodiment, synthetic nanocarriers according to the invention that havea minimum dimension of equal to or less than about 100 nm, preferablyequal to or less than 100 nm, do not comprise a surface that activatescomplement or alternatively comprise a surface that consists essentiallyof moieties that do not activate complement. In embodiments, syntheticnanocarriers exclude virus-like particles. In embodiments, syntheticnanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5,1:2, 1:3, 1:5, 1:7, or greater than 1:10.

“Treating” refers to the administration of one or more therapeutics withthe expectation that the subject may have a resulting benefit due to theadministration. The treating may also result in the prevention of acondition as provided herein and, therefore, treating includesprophylactic treatment. When used prophylactically, the subject is onein which a clinician expects that there is a likelihood for thedevelopment of a condition or other undesired response as providedherein. In some embodiments, a subject that is expected to have a goutflare is one in which a clinician believes there is a likelihood that agout flare will occur. Treating may be direct or indirect, such as byinducing or directing another subject, including another clinician orthe subject itself, to treat the subject.

“Weight%” or “% by weight” refers to the ratio of one weight to anotherweight times 100. For example, the weight% can be the ratio of theweight of one component to another times 100 or the ratio of the weightof one component to a total weight of more than one component times 100.Generally, the weight% is measured as an average across a population ofsynthetic nanocarriers or an average across the synthetic nanocarriersin a composition or suspension.

C. Methods and Related Compositions

As mentioned elsewhere herein, it has been demonstrated that thecompositions and methods provided herein are substantially moreefficacious at reducing serum uric acid levels as well as reducing thefrequency of gout flares than currently available treatments.

Uricase and Pegylated Uricase

The methods and compositions and kits described herein involvecompositions comprising uricase. Uricase is generally thought tocatalyze the conversion of uric acid to allantoin, which is soluble andmay be excreted. Uricase generally is an enzyme endogenous to allmammals, except for humans and certain primates. The gene encoding theuricase enyzme may be obtained from any source known in the art,including mammalian and microbial sources as well as by recombinant andsynthetic technologies. As will be evident to one of ordinary skill inthe art, a gene may be obtained from a source and recombinantly (ortransgenically) expressed and produced in another organism usingstandard methods. See Erlich, H A, (Ed.) (1989) PCR Technology.Principles and Applications for DNA Amplification. New York: StocktonPress; Sambrook, J, et al., (1989) Molecular Cloning. A LaboratoryManual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press. For example, U.S. Pat. No. 5,700,674 describesrecombinant production of uricase in E. coli cells. In some embodiments,the enzyme is produced by fermentation in E. coli.

In some embodiments, the gene encoding the uricase, or a portionthereof, is obtained from a mammal, for example a pig, bovine, sheep,goat, baboon, monkey mouse, rabbit, or domestic animal. In someembodiments, the gene encoding the uricase, or a portion thereof, isobtained from a microorganism, such as a bacteria or fungi (includingyeast). In some embodiments, the gene encoding the uricase is obtainedfrom a bacterial source, such as bacterium belonging to Streptomycesspp., Bacillus spp., or E. coli. In some embodiments, the gene encodingthe uricase is obtained from a fungal (including yeast) source, such asCandida (e.g., Candida utilis), Anthrobacter (e.g., Anthrobacterglobiformis), Saccharomyces, Schizosaccaromyces, Emericella, Aspergillus(e.g., Aspergillus flavus), and Neurospora spp. In some embodiments, theuricase is derived from Candida utilis. In some embodiments, the uricaseis that of pegsiticase (3SBio as described in U.S. Pat. No. 6,913,915,and such uricase and description thereof is incorporated herein byreference). In some embodiments, the uricase is derived from Aspergillusflavus. In some embodiments, the uricase is rasburicase (ELITEK®;FASTURTEC®, from Sanofi Genzyme).

In some embodiments, the uricase is chimeric uricase, in which portionsof the gene encoding the uricase are obtained from different sources.For example, a portion of the gene encoding the chimeric uricase may beobtained from one organism and one or more other portions of the geneencoding the chimeric uricase may be obtained from another organism. Insome embodiments, a portion of the gene encoding the chimeric uricase isobtained from a pig and another portion of the gene encoding thechimeric uricase is obtained from a baboon. In some embodiments, thechimeric uricase is that of pegloticase/KRYSTEXXA®.

Also within the scope of the present invention are variant uricases,which may include one or more mutations (substitutions, insertions,deletions). Mutations may be made in the nucleotide sequence encodingthe uricase protein, which may or may not result in an amino acidmutation. In general, mutations may be made, for example, to enhanceproduction of the protein, turnover/half-life of the protein or mRNAencoding the protein, modulate (enhance or reduce) the enzymaticactivity of the uricase.

In other embodiments, the gene encoding the uricase is obtained from aplant or invertebrate source, such as Drosophila or C. elegans.

Any of the uricase proteins described herein may be pegylated. Uricasemay be covalently bonded to PEG via a biocompatible linking group, usingmethods known in the art, as described, for example, by Park et al,Anticancer Res., 1:373-376 (1981); and Zaplipsky and Lee, PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, ed., Plenum Press, New York, Chapter 21 (1992). The linkinggroup used to covalently attach PEG to uricase may be any biocompatiblelinking group, meaning the linking group non-toxic and may be utilizedin vitro or in vivo without causing adverse effects. Alternatively, thePEG may be directly conjugated to the uricase, such as directly to alysine residue of uricase.

Uricase may be pegylated at many different amino acid resides of theuricase protein. The number of PEG molecules and/or residue to which thePEG is conjugated may affect the activity of the uricase. In someembodiments, the pegylated uricase comprises at least one PEG molecule.In some embodiments, the pegylated uricase comprises at least 2, 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 45,50, or more PEG molecules on average per uricase protein. In someembodiments, the pegylated uricase comprises about 20-25 PEG moleculesper uricase protein.

On average, PEG has a molecular weight between 5 kDa to 100 kDa. Boththe molecular weight (size) of the PEG used as well as the number of PEGmolecules used to pegylate the uricase may be varied. In some embodimentthe average molecular weight of the PEG is between 5 kDa to 100 kDa, 5kDa to 75 kDa, 5 kDa to 50 kDa, 5 kDa to 30 kDa, 5 kDa to 20 kDa, 5 kDato 10 kDa, 10 kDa to 75 kDa, 10 kDa to 50 kDa, 10 kDa to 30 kDa, 5 kDato 30 kDa, 15 kDa to 50 kDa, 15 kDa to 30 kDa, 15 kDa to 25 kDa, 20 kDato 75 kDa, 30 kDa to 80 kDa, 30 kDa to 70 kDa, or 30 kDa to 50 kDa. Insome embodiments, the molecular weight of the PEG is about 5 kDa, 6 kDa,7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, or 100 kDa. In general, thePEG is referred to based on the molecular weight of the PEG. Forexample, PEG-20 refers to PEG molecules with a molecular weight of 20kDa, and PEG-5 refers to PEG molecules with a molecular weight of 5 kDa.In some embodiments, the uricase is pegylated with PEG molecules havinga molecule weight of 20 kDa (PEG-20).

Pegylated uricases include, without limitation, pegsiticase (i.e.,pegadricase) (available from 3Sbio, and as described in U.S. Pat. No.6,913,915, and such pegylated uricase and description thereof isincorporated herein by reference) and pegloticase/KRYSTEXXA® (HorizonPharmaceuticals).

Preferably, in some embodiments of any one of the methods orcompositions or kits provided herein, the pegylated uricase ispegsiticase (i.e., pegadricase), a recombinant uricase conjugated tomultiple 20 kDa molecular weight poly (ethylene glycol) molecules. Theuricase component of pegsiticase can be cloned from the yeast Candidautilis and expressed in E. coli for production.

The uric acid catalysis activity of uricase, including pegylateduricase, can be assessed using methods known in the art or as otherwiseprovided herein.

Synthetic Nanocarriers

A variety of synthetic nanocarriers can be used. In some embodiments,synthetic nanocarriers are spheres or spheroids. In some embodiments,synthetic nanocarriers are flat or plate-shaped. In some embodiments,synthetic nanocarriers are cubes or cubic. In some embodiments,synthetic nanocarriers are ovals or ellipses. In some embodiments,synthetic nanocarriers are cylinders, cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size or shape sothat each synthetic nanocarrier has similar properties. For example, atleast 80%, at least 90%, or at least 95% of the synthetic nanocarriers,based on the total number of synthetic nanocarriers, may have a minimumdimension or maximum dimension that falls within 5%, 10%, or 20% of theaverage diameter or average dimension of the synthetic nanocarriers.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. Polymers may be natural or unnatural (synthetic)polymers. Polymers may be homopolymers or copolymers comprising two ormore monomers. In terms of sequence, copolymers may be random, block, orcomprise a combination of random and block sequences. Typically,polymers in accordance with the present invention are organic polymers.

The synthetic nanocarriers as provided herein, preferably, comprisehydrophobic polyesters. Such polyesters can include copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, the polyester may be PLGA. PLGA is a biocompatibleand biodegradable co-polymer of lactic acid and glycolic acid, andvarious forms of PLGA are characterized by the ratio of lacticacid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, orD,L-lactic acid. The degradation rate of PLGA can be adjusted byaltering the lactic acid:glycolic acid ratio. In some embodiments, PLGAto be used in accordance with the present invention is characterized bya lactic acid:glycolic acid ratio of approximately 85:15, approximately75:25, approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

The synthetic nanocarriers may comprise one or more non-polyesterpolymers or units thereof that are also hydrophobic and/or polymers orunits thereof that are not hydrophobic. In some embodiments, it ispreferred that overall the synthetic nanocarrier comprises a hydrophobicpolyester and, in some embodiments, is itself hydrophobic.

The synthetic nanocarriers may comprise one or more polymers that are anon-methoxy-terminated, pluronic polymer, or a unit thereof.“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini.

The synthetic nanocarriers may comprise, in some embodiments,polyhydroxyalkanoates, polyamides, polyethers, polyolefins,polyacrylates, polycarbonates, polystyrene, silicones, fluoropolymers,or a unit thereof. Further examples of polymers that may be comprised inthe synthetic nanocarriers provided herein include polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymers of the synthetic nanocarriers may comprise poly(ethyleneglycol) (PEG), polypropylene glycol, or unit thereof.

In some embodiments, it is preferred that the synthetic nanocarrierscomprise polymer that is biodegradable. Therefore, in such embodiments,the polymers of the synthetic nanocarriers may include a polyether, suchas poly(ethylene glycol) or polypropylene glycol or unit thereof.Additionally, the polymer may comprise a block-co-polymer of a polyetherand a biodegradable polymer such that the polymer is biodegradable. Inother embodiments, the polymer does not solely comprise a polyether orunit thereof, such as poly(ethylene glycol) or polypropylene glycol orunit thereof.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600.

Other examples of polymers suitable for use in synthetic nanocarriersinclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(P-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

Still other examples of polymers that may be included in the syntheticnanocarriers include acrylic polymers, for example, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, aminoalkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acidalkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acidanhydride), methyl methacrylate, polymethacrylate, poly(methylmethacrylate) copolymer, polyacrylamide, aminoalkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. A wide variety of polymers and methods forforming polymeric matrices therefrom are known conventionally. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polyesterhas a hydrophobic environment within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5543158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that the synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention provided they meet the desired criteria.

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pats. 6,123,727;5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752;5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379;5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J.Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460;Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release,62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, avariety of methods for synthesizing certain suitable polymers aredescribed in Concise Encyclopedia of Polymer Science and PolymericAmines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980;Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pats. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods such as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling (including cryomilling),supercritical fluid (such as supercritical carbon dioxide) processing,microemulsion procedures, microfabrication, nanofabrication, sacrificiallayers, simple and complex coacervation, and other methods well known tothose of ordinary skill in the art. Alternatively or additionally,aqueous and organic solvent syntheses for monodisperse semiconductor,conductive, magnetic, organic, and other nanomaterials have beendescribed (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000,Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat.,13:3843). Additional methods have been described in the literature (see,e.g., Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine andPharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J.Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers,6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.Pats. 5578325 and 6007845; P. Paolicelli et al., “Surface-modifiedPLGA-based Nanoparticles that can Efficiently Associate and DeliverVirus-like Particles” Nanomedicine. 5(6):843-853 (2010)).

Immunosuppressants may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8- 21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. 6,632,671 to Unger issued Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe included in the synthetic nanocarriers and/or the composition of thecarrier matrix.

If synthetic nanocarriers prepared by any of the above methods have asize range outside of the desired range, such synthetic nanocarriers canbe sized, for example, using a sieve.

Preferably, in some embodiments of any one of the methods orcompositions or kits provided herein, the synthetic nanocarriers arethose that comprise synthetic nanocarriers composed of PLA and PLA-PEG.PLA is part of the broader poly(lactic co glycolic acid), or PLGA,family of biodegradable polymers that have more than 30 years ofcommercial use and are formulation components in a number of approvedproducts. Polyethylene glycol, or PEG, has been widely studied inclinical trials and is also a formulation component in many approvedbiologic products.

As examples, the synthetic nanocarriers comprising rapamycin can bethose produced or obtainable by one of the following methods:

-   1) PLA with an inherent viscosity of 0.41 dL/g is purchased from    Evonik Industries (Rellinghauser Straße 1—11 45128 Essen, Germany),    product code Resomer Select 100 DL 4A. PLA-PEG-OMe block co-polymer    with a methyl ether terminated PEG block of approximately 5,000 Da    and an overall inherent viscosity of 0.50 DL/g is purchased from    Evonik Industries (Rellinghauser StraBe 1—11 45128 Essen, Germany),    product code Resomer Select 100 DL mPEG 5000 (15 wt% PEG). Rapamycin    is purchased from Concord Biotech Limited (1482-1486 Trasad Road,    Dholka 382225, Ahmedabad India), product code SIROLIMUS. EMPROVE®    Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6    mPa.s) is purchased from MilliporeSigma (EMD Millipore, 290 Concord    Road Billerica, Massachusetts 01821), product code 1.41350.    Dulbecco’s phosphate buffered saline 1X (DPBS) is purchased from    Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland),    product code 17-512Q. Sorbitan monopalmitate is purchased from Croda    International (300-A Columbus Circle, Edison, NJ 08837), product    code SPAN 40. Solutions are prepared as follows. Solution 1 is    prepared by dissolving PLA at 150 mg/mL and PLA-PEG-Ome at 50 mg/mL    in dichloromethane. Solution 2 is prepared by dissolving rapamycin    at 100 mg/mL in dichloromethane. Solution 3 is prepared by    dissolving SPAN 40 at 50 mg/mL in dichloromethane. Solution 4 is    prepared by dissolving PVA at 75 mg/mL in 100 mM phosphate buffer    pH 8. O/W emulsions are prepared by adding Solution 1 (0.50 mL),    Solution 2 (0.12 mL), Solution 3 (0.10 mL), and dichloromethane    (0.28 mL), in a thick walled glass pressure tube. The combined    organic phase solutions are then mixed by repeat pipetting. To this    mixture, Solution 4 (3 mL), is added. The pressure tube is then    vortex mixed for 10 seconds. Next, the crude emulsion is homogenized    by sonication at 30% amplitude for 1 minute using a Branson Digital    Sonifier 250 with a ⅛″ tapered tip, and the pressure tube immersed    in an ice water bath. The emulsion is then added to a 50 mL beaker    containing DPBS (30 mL). This is stirred at room temperature for 2    hours to allow the dichloromethane to evaporate and for the    nanocarriers to form. A portion of the nanocarriers is washed by    transferring the nanocarrier suspension to a centrifuge tube and    centrifuging at 75,600×g at 4° C. for 50 minutes, removing the    supernatant, and re-suspended the pellet in DPBS containing 0.25%    w/v PVA. The wash procedure is repeated and the pellet is    re-suspended in DPBS containing 0.25% w/v PVA to achieve a    nanocarrier suspension having a nominal concentration of 10 mg/mL on    a polymer basis. The nanocarrier suspension is then filtered using a    0.22 µm PES membrane syringe filter from MilliporeSigma (EMD    Millipore, 290 Concord Rd. Billerica MA, product code SLGP033RB).    The filtered nanocarrier suspension is stored at -20° C.-   2) PLA with an inherent viscosity of 0.41 dL/g is purchased from    Evonik Industries (Rellinghauser StraBe 1—11 45128 Essen, Germany),    product code Resomer Select 100 DL 4A. PLA-PEG-OMe block co-polymer    with a methyl ether terminated PEG block of approximately 5,000 Da    and an overall inherent viscosity of 0.50 DL/g is purchased from    Evonik Industries (Rellinghauser StraBe 1—11 45128 Essen, Germany),    product code Resomer Select 100 DL mPEG 5000 (15 wt% PEG). Rapamycin    is purchased from Concord Biotech Limited (1482-1486 Trasad Road,    Dholka 382225, Ahmedabad India), product code SIROLIMUS. Sorbitan    monopalmitate is purchased from Sigma-Aldrich (3050 Spruce St., St.    Louis, MO 63103), product code 388920. EMPROVE® Polyvinyl Alcohol    (PVA) 4-88, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa. s) is    purchased from MilliporeSigma (EMD Millipore, 290 Concord Road    Billerica, Massachusetts 01821), product code 1.41350. Dulbecco’s    phosphate buffered saline 1X (DPBS) is purchased from Lonza    (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland), product    code 17-512Q. Solutions are prepared as follows: Solution 1: A    polymer, rapamycin, and sorbitan monopalmitate mixture is prepared    by dissolving PLA at 37.5 mg/mL, PLA-PEG-Ome at 12.5 mg/mL,    rapamycin at 8 mg/mL, and sorbitan monopalmitate at 2.5 in    dichloromethane. Solution 2: Polyvinyl alcohol is prepared at 50    mg/mL in 100 mM pH 8 phosphate buffer. An O/W emulsion is prepared    by combining Solution 1 (1.0 mL) and Solution 2 (3 mL) in a small    glass pressure tube, and vortex mixed for 10 seconds. The    formulation is then homogenized by sonication at 30% amplitude for 1    minute using a Branson Digital Sonifier 250 with a ⅛″ tapered tip,    with the pressure tube immersed in an ice water bath. The emulsion    is then added to a 50 mL beaker containing DPBS (15 mL), and covered    with aluminum foil. A second O/W emulsion is prepared using the same    materials and method as above and then added to the same beaker    using a fresh aliquot of DPBS (15 mL). The combined emulsion is then    left uncovered and stirred at room temperature for 2 hours to allow    the dichloromethane to evaporate and for the nanocarriers to form. A    portion of the nanocarriers is washed by transferring the    nanocarrier suspension to a centrifuge tube and centrifuging at    75,600×g and 4° C. for 50 minutes, removing the supernatant, and    re-suspending the pellet in DPBS containing 0.25% w/v PVA. The wash    procedure is repeated and then the pellet re-suspended in DPBS    containing 0.25% w/v PVA to achieve a nanocarrier suspension having    a nominal concentration of 10 mg/mL on a polymer basis. The    nanocarrier suspension is then filtered using a 0.22 µm PES membrane    syringe filter from MilliporeSigma (EMD Millipore, 290 Concord Rd.    Billerica MA, product code SLGP033RB). The filtered nanocarrier    suspension is then stored at -20° C.

Immunosuppressants

Any immunosuppressant as provided herein can be used in any one of themethods or compositions provided and can be, in some embodiments,attached to synthetic nanocarriers. Immunosuppressants include, but arenot limited to, mTOR inhibitors. Examples of mTOR inhibitors includerapamycin and rapalogs (e.g., CCL-779, RAD001, AP23573,C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamidorapamycin(C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al.Chemistry & Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235),chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus(RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354(available from Selleck, Houston, TX, USA).

Preferably, in some embodiments of any one of the methods orcompositions or kits provided herein, the immunosuppressant israpamycin. In some of such embodiments, the rapamycin is preferablyencapsulated in the synthetic nanocarriers. Rapamycin is the activeingredient of Rapamune, an immunosuppressant which has extensive prioruse in humans and is currently FDA approved for prophylaxis of organrejection in kidney transplant patients aged 13 or older.

When coupled to a synthetic nanocarrier, the amount of theimmunosuppressant coupled to the synthetic nanocarrier based on thetotal dry recipe weight of materials in an entire synthetic nanocarrier(weight/weight), is as described elsewhere herein. Preferably, in someembodiments of any one of the methods or compositions or kits providedherein, the load of the immunosuppressant, such as rapamycin or rapalog,is between 7% and 12% or 8% and 12% by weight.

Anti-Inflammatory Therapeutics

Anti-inflammatory therapeutics (i.e., any therapeutic that can act toreduce inflammation). Anti-inflammatory therapeutics include, but arenot limited to, corticosteroids or derivatives of cortisol(hydrocortisone). Corticosteroids include, but are not limited to,glucocorticoids and mineralocorticoids. Still other examples ofcorticosteroids include, but are not limited to, those that are natural(e.g., 11-Dehydrocorticosterone (11-oxocorticosterone,17-deoxycortisone) = 21-hydroxypregn-4-ene-3,11,20-trione;11-Deoxycorticosterone (deoxycortone, desoxycortone;21-hydroxyprogesterone) = 21-hydroxypregn-4-ene-3,20-dione;11-Deoxycortisol (cortodoxone, cortexolone) =17α,21-dihydroxypregn-4-ene-3,20-dione; 11-Ketoprogesterone(11-oxoprogesterone; Ketogestin) = pregn-4-ene-3,11,20-trione;11β-Hydroxypregnenolone = 3β,11β-dihydroxypregn-5-en-20-one;11β-Hydroxyprogesterone (21-deoxycorticosterone) =11β-hydroxypregn-4-ene-3,20-dione; 11β,17α,21-Trihydroxypregnenolone =3β,11β,17α,21-tetrahydroxypregn-5-en-20-one;17α,21-Dihydroxypregnenolone = 3β3,17α,21-trihydroxypregn-5-en-20-one;17α-Hydroxypregnenolone = 3β,17α-dihydroxypregn-5-en-20-one;17α-Hydroxyprogesterone = 17α-hydroxypregn-4-ene-3,11,20-trione;18-Hydroxy-11-deoxycorticosterone =18,21-dihydroxypregn-4-ene-3,20-dione; 18-Hydroxycorticosterone =11β,18,21-trihydroxypregn-4-ene-3,20-dione; 18-Hydroxyprogesterone =18-hydroxypregn-4-ene-3,20-dione; 21-Deoxycortisol =11β,17α-dihydroxypregn-4-ene-3,20-dione; 21-Deoxycortisone =17α-hydroxypregn-4-ene-3,11,20-trione; 21-Hydroxypregnenolone(prebediolone) = 3β,21-dihydroxypregn-5-en-20-one; Aldosterone =11β,21-dihydroxypregn-4-ene-3,18,20-trione; Corticosterone(17-deoxycortisol) = 11β,21-dihydroxypregn-4-ene-3,20-dione; Cortisol(hydrocortisone) = 11β,17α,21-trihydroxypregn-4-ene-3,20-dione;Cortisone = 17α,21-dihydroxypregn-4-ene-3,11,20-trione; Pregnenolone =pregn-5-en-3β-ol-20-one; and Progesterone = pregn-4-ene-3,20-dione);those that are synthetic, such as progesterone-type (e.g., Flugestone(flurogestone) = 9α-fluoro-11β,17α-dihydroxypregn-4-ene-3,20-dione;Fluorometholone = 6α-methyl-9α-fluoro-11β,17α-dihydroxypregna-1,4-diene-3,20-dione; Medrysone(hydroxymethylprogesterone) = 6α-methyl-β-hydroxypregn-4-ene-3,20-dione;and Prebediolone acetate (21-acetoxypregnenolone) =3β,21-dihydroxypregn-5-en-20-one 21-acetate) and progesterone derivativeprogestins (e.g., chlormadinone acetate, cyproterone acetate,medrogestone, medroxyprogesterone acetate, megestrol acetate, andsegesterone acetate); hydrocortisone-type (e.g., Chloroprednisone =6α-chloro-17α,21-dihydroxypregna-1,4-diene-3,11,20-trione; Cloprednol =6-chloro-11β,17α,21-trihydroxypregna-1,4,6-triene-3,20-dione;Difluprednate =6α,9α-difluoro-11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione17α-butyrate 21-acetate; Fludrocortisone =9α-fluoro-11β,17α,21-trihydroxypregn-4-ene-3,20-dione; Fluocinolone =6α,9α-difluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione;Fluperolone =9α-fluoro-11β,17α,21-trihydroxy-21-methylpregna-1,4-diene-3,20-dione;Fluprednisolone =6α-fluoro-11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione; Loteprednol= 11β,17α,dihydroxy-21-oxa-21-chloromethylpregna-1,4-diene-3,20-dione;Methylprednisolone =6α-methyl-11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione;Prednicarbate = 11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione17α-ethylcarbonate 21-propionate; Prednisolone =11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione; Prednisone =17α,21-dihydroxypregna-1,4-diene-3,11,20-trione; Tixocortol =11β,17α-dihydroxy-21-sulfanylpregn-4-ene-3,20-dione; and Triamcinolone =9α-fluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione);methasone-type (16-methylated) (e.g., Methasone; Alclometasone =7α-chloro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Beclometasone =9α-chloro-11β,17α,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione;Betamethasone = 9α-fluoro- 1 1β, 17α,2 1 -trihydroxy-16β-methylpregna- 1,4-diene-3,20-dione; Clobetasol =9α-fluoro-11β,17α-dihydroxy-16β-methyl-21-chloropregna-1,4-diene-3,20-dione;Clobetasone =9α-fluoro-16β-methyl-17α-hydroxy-21-chloropregna-1,4-diene-3,11,20-trione;Clocortolone =6α-fluoro-9α-chloro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Desoximetasone =9α-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Dexamethasone =9α-fluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Diflorasone =6α,9α-difluoro-11β,17α,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione;Difluocortolone = 6α,9α-difluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione; Fluclorolone =6α-fluoro-9α,11β-dichloro-16α,17α,21-trihydroxypregna-1,4-dien-3,20-dione;Flumetasone =6α,9α-difluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Fluocortin =6α-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20,21-trione;Fluocortolone =6α-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione;Fluprednidene =9α-fluoro-11β,17α,21-trihydroxy-16-methylenepregna-1,4-diene-3,20-dione;Fluticasone =6α,9α-difluoro-11β,17α-dihydroxy-16α-methyl-21-thia-21-fluoromethylpregna-1,4-dien-3,20-dione;Fluticasone furoate =6α,9α-difluoro-11β,17a-dihydroxy-16a-methyl-21-thia-21-fluoromethylpregna-1,4-dien-3,20-dione17a-(2-furoate); Halometasone =2-chloro-6a,9a-difluoro-11β,17a,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione;Meprednisone =16β-methyl-17a,21-dihydroxypregna-1,4-diene-3,11,20-trione; Mometasone =9a,21-dichloro-11β,17a-dihydroxy-16a-methylpregna-1,4-diene-3,20-dione;Mometasone furoate =9a,21-dichloro-11β,17a-dihydroxy-16a-methylpregna-1,4-diene-3,20-dione17a-(2-furoate); Paramethasone = 6a-fluoro-l 11β,17a,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione; Prednylidene =11β,17a,21-trihydroxy-16-methylenepregna-1,4-diene-3,20-dione;Rimexolone = 11β-hydroxy-16a,17a,21-trimethylpregna-1,4-dien-3,20-dione;and Ulobetasol (halobetasol) =6a,9a-difluoro-11β,17a-dihydroxy-16β-methyl-21-chloropregna-1,4-diene-3,20-dione);Acetonides and related (e.g., Amcinonide =9a-fluoro-11β,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16a,17a-acetal with cyclopentanone, 21-acetate; Budesonide =11β,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16a,17a-acetal with butyraldehyde; Ciclesonide =11β,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16a,17a-acetal with (R)-cyclohexanecarboxaldehyde, 21-isobutyrate;Deflazacort =11β,21-dihydroxy-2′-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione21-acetate; Desonide =11β,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16a,17a-acetal with acetone; Formocortal (fluoroformylone) =3-(2-chloroethoxy)-9a-fluoro-11β,16a,17a,21-tetrahydroxy-20-oxopregna-3,5-diene-6-carboxaldehydecyclic 16a,17a-acetal with acetone, 21-acetate; Fluclorolone acetonide(flucloronide) =6a-fluoro-9a,11β-dichloro-16α,17α,21-trihydroxypregna-1,4-dien-3,20-dionecyclic 16a,17a-acetal with acetone; Fludroxycortide (flurandrenolone,flurandrenolide) =6α-fluoro-11β,16α,17α,21-tetrahydroxypregn-4-ene-3,20-dione cyclic16a,17a-acetal with acetone; Flunisolide =6α-fluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16a,17a-acetal with acetone; Fluocinolone acetonide =6α,9α-difluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dionecyclic 16α,17α-acetal with acetone; Fluocinonide =6α,9α-difluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dionecyclic 16α,17α-acetal with acetone, 21-acetate; Halcinonide =9α-fluoro-11β,16α,17α-trihydroxy-21-chloropregn-4-ene-3,20-dione cyclic16α,17α-acetal with acetone; and Triamcinolone acetonide =9α-fluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic16α,17α-acetal with acetone); and still others (e.g., Cortivazol =6,16α-dimethyl-11β,17α,21-trihydroxy-2′-phenyl[3,2-c]pyrazolopregna-4,6-dien-20-one21-acetate; and RU-28362 =6-methyl-11β,17β-dihydroxy-17α-(1-propynyl)androsta-1,4,6-trien-3-one).

Corticosteroids, particularly glycocorticoids, have anti-inflammatoryand immunosuppressive effects that may be effective in managingsymptoms, including pain and inflammation associated with gout, goutflare, and/or conditions associated with gout. Administration ofcorticosteroids may also aid in reducing hypersensitivity reactionsassociated with one or more additional therapies, for example uricasereplacement therapy. Still other non-limiting examples ofcorticosteroids, include prednisone, prednisolone, Medrol, andmethylprednisolone.

Infusion Reaction Therapeutic

Infusion reaction therapeutics (i.e., any therapeutic that can bebeneficial in reducing or preventing infusion reactions) can also beincluded in the compositions and related methods provided herein. Suchtherapeutics can include an anti-inflammatory, such as any one of theanti-inflammatories provided herein (e.g., a corticosteroid, such asmethylprednisolone, prednisone or dexamethasone). Such therapeutics canalso include an antihistamine. Antihistamines are agents that caninhibit the physiological effects of histamine, and includeBrompheniramine, Carbinoxamine, Chlorpheniramine, Clemastine,Diphenhydramine, Hydroxyzine, Triprolidine, Cetirizine, Desloratadine,Fexofenadine, Levocetirizine, Loratadine, etc.

Dosing

Unless otherwise specified herein, the amount (by weight) of a dose of acomposition comprising pegylated uricase as well as the concentrationsper vial provided herein refers to the amount or concentration of theuricase protein, respectively, not including the PEG moleculesconjugated thereto or any added excipients in the composition. Theactual amount of the pegylated uricase, in such instances, will behigher than the dose described due to the higher weight of the pegylatedprotein form. In one example, a dose of 0.4 mg/kg of a compositioncomprising pegylated uricase refers to a dose of 0.4 mg/kg uricaseprotein.

Thus, a dose of a composition comprising pegylated uricase foradministration to a subject may be calculated based on the dose providedherein and the weight of the subject, according to the followingequation:

(dose in mg/kg (this is of the uricase protein)) x (subject weight (kg))/ (concentration per mL in vial (again this is of the uricase protein))= volume to be administered

As an example, the pegylated uricase may be reconstituted in sterilewater to a concentration of 6 mg/mL. Thus, for this example, for a doseof 0.4 mg/kg to be administered to a subject weighing 90.7 kg (200 lbs),6.048 mL of the reconstituted pegylated uricase composition should beadministered to the subject:

(0.4 mg/kg) x (90.7 kg) / (6 mg/mL) = 6.048 mL

In some embodiments, the appropriate volume of the compositioncomprising pegylated uricase is diluted in a pharmaceutically acceptableexcipient (e.g., sterile saline solution) for, for example, intravenousinfusion to a subject over a desired period of time (e.g., 60 minutes).

Similarly, unless otherwise specified herein, the amount (by weight) ofa dose of a composition comprising synthetic nanocarriers comprising animmunosuppressant as well as the concentrations per vial as providedherein refers to the amount or concentration of the immunosuppressant,respectively, and not including the synthetic nanocarrier material orany added excipients or other components in the composition. The actualamount of the synthetic nanocarrier composition comprising theimmunosuppressant will be higher than the dose described due to theadded weight of the synthetic nanocarrier material and any addedexcipients or other components in the composition. In one example, adose of 0.08 mg/kg of a composition comprising synthetic nanocarrierscomprising an immunosuppressant refers to a dose of 0.08 mg/kgimmunosuppressant.

Thus, a dose of a composition comprising synthetic nanocarrierscomprising an immunosuppressant for administration to a subject may becalculated based on the weight of the subject, according to thefollowing equation:

(dose in mg/kg (this is of the immunosuppressant)) x (subject weight(kg)) / (concentration per mL in vial (again this is of theimmunosuppressant) = volume to be administered

As an example, the composition comprising synthetic nanocarrierscomprising an immunosuppressant is at a concentration of 2 mg/mL (againthis is the concentration of the immunosuppressant). Thus, for thisexample, for a dose of 0.08 mg/kg to be administered to a subjectweighing 90.7 kg (200 lbs), 3.6 mL of the composition should beadministered to the subject:

(0.08 mg/kg) x (90.7 kg) / (2 mg/mL) = 3.6 mL

The load of the immunosuppressant (e.g., rapamycin) of the syntheticnanocarriers comprising an immunosuppresant may be determined byextracting the immunosuppressant from the synthetic nanocarriers usingliquid extraction compatible with both the immunosuppressant and thesynthetic nanocarriers (e.g., polymers comprising the syntheticnanocarriers) and analyzing the extract by reverse phase liquidchromatography with UV detection specific for the analyte. Theimmunosuppressant load (content of the synthetic nanocarriers) may beaccurately and precisely calculated from a calibration standard curve ofa qualified reference standard prepared in conditions compatible withthe chromatography and the nanoparticle extraction procedure andanalyzed concomitantly.

The amount (by weight) of a dose of a composition comprising syntheticnanocarriers comprising an immunosuppressant may be calculated based onthe amount (by weight) of the immunosuppressant dose, according to thefollowing equation:

(⅟load of immunosuppressant) x (dose given based on the amount ofimmunosuppressant) = dose of immunosuppressant given as the amount ofthe synthetic nanocarriers comprising the immunosuppressant

As an example, the load of immunosuppressant in the syntheticnanocarriers can be about 10% and if a dose of 0.08 mg/kg of theimmunosuppressant is desired, the dose given as the amount of thesynthetic nanocarriers comprising the immunosuppressant is 8 mg/kg.

The amount of uricase protein present in a pegylated uricase may bedetermined using methods known in the art, for example colorimetry, UVabsorbance or amino acid analysis. The colorimetric approach relies on astandardized kit commercially available leveraging typical dye basedreactions such as those described for Bradford or bicinchoninic acid(BCA) assays. The uricase protein quantity can be accurately andprecisely calculated from a calibration standard curve of a qualifiedprotein reference standard, preferably purchased from compendialsources, and analyzed concomitantly using the same spectrophotometer.Single or multiple point calibration of a known protein of similar ordifferent chemical properties may be run within the same assay to ensureconsistency of the read out at the chosen UV absorbance. The amino acidmixture obtained from acid hydrolysis of the drug product may also beanalyzed and generally provides a precise and accurate quantification.The amino acid mixture is analyzed by HPLC with either UV orfluorescence detection and using pre-chromatography orpost-chromatography derivatization of the primary and secondary amines.Commercially available mixtures of common amino acids are analyzedwithin the same assay to build the individual amino acid calibrationcurves against which each amino acid is quantified. In some embodiments,the determination of the uricase protein quantity is supplemented bymeasuring the enzyme activity, which may be performed by measuring thedecrease of an excess of uric acid monitored by UV absorbance at 595 nm.Alternatively or in addition, the uricase activity can be determinedusing a commercially available kit, which may involve, for example,labeling the enzymatic reaction product and measuring the response ofthe uricase against a calibration curve established by analyzing a knownquantity of the enzyme.

Similar to the immediately above formula, the amount (by weight) of adose of a composition comprising pegylated uricase can be calculatedbased on the amount (by weight) of the uricase dose, according to thefollowing equation:

(⅟(weight of uricase of a pegylated uricase/weight of the pegylateduricase)) x (dose given based on the amount of uricase) = dose ofpegylated uricase given as the amount of the pegylated uricase

It should be understood that the amount provided herein can be anaverage amount based on a population of the respective molecules in acomposition.

Exemplary doses of uricase for any one of the compositions or methodscomprising uricase, such as pegsiticase (i.e., pegadricase), as providedherein can be 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, 0.20mg/kg, 0.21 mg/kg, 0.22 mg/kg, 0.23 mg/kg, 0.24 mg/kg, 0.25 mg/kg, 0.26mg/kg, 0.27 mg/kg, 0.28 mg/kg, 0.29 mg/kg, 0.30 mg/kg, 0.31 mg/kg, 0.32mg/kg, 0.34 mg/kg, 0.35 mg/kg, 0.36 mg/kg, 0.37 mg/kg, 0.38 mg/kg, 0.39mg/kg, 0.40 mg/kg, 0.41 mg/kg, 0.42 mg/kg, 0.43 mg/kg, 0.44 mg/kg, 0.45mg/kg, 0.46 mg/kg, 0.47 mg/kg, 0.48 mg/kg, 0.49 mg/kg, 0.50 mg/kg, 0.51mg/kg, 0.52 mg/kg, 0.53 mg/kg, 0.54 mg/kg, 0.55 mg/kg, 0.56 mg/kg, 0.57mg/kg, 0.58 mg/kg, 0.59 mg/kg, 0.60 mg/kg, 0.61 mg/kg, 0.62 mg/kg, 0.63mg/kg,0.64 mg/kg, 0.65 mg/kg, 0.66 mg/kg, 0.67 mg/kg, 0.68 mg/kg, 0.69mg/kg, 0.70 mg/kg, 0.71 mg/kg, 0.72 mg/kg, 0.73 mg/kg, 0.74 mg/kg, 0.75mg/kg, 0.76 mg/kg, 0.77 mg/kg, 0.78 mg/kg, 0.79 mg/kg, 0.80 mg/kg, 0.81mg/kg, 0.82 mg/kg, 0.83 mg/kg, 0.84 mg/kg, 0.85 mg/kg, 0.86 mg/kg, 0.87mg/kg, 0.88 mg/kg, 0.89 mg/kg, 0.90 mg/kg, 0.91 mg/kg, 0.92 mg/kg, 0.93mg/kg, 0.94 mg/kg, 0.95 mg/kg, 0.96 mg/kg, 0.97 mg/kg, 0.98 mg/kg, 0.90mg/kg, 1.0 mg/kg, 1.01 mg/kg, 1.02 mg/kg, 1.03 mg/kg, 1.04 mg/kg, 1.05mg/kg, 1.06 mg/kg, 1.07 mg/kg, 1.08 mg/kg, 1.09 mg/kg, 1.10 mg/kg, 1.11mg/kg, 1.12 mg/kg, 1.13 mg/kg, 1.14 mg/kg, 1.15 mg/kg, 1.16 mg/kg, 1.17mg/kg, 1.18 mg/kg, 1.19 mg/kg, or 1.20 mg/kg uricase.

Exemplary doses of immunosuppressant, such as rapamycin, for any one ofthe compositions or methods comprising synthetic nanocarriers comprisingthe immunosuppressant provided herein can be 0.050 mg/kg, 0.055 mg/kg,0.060 mg/kg, 0.065 mg/kg, 0.070 mg/kg, 0.075 mg/kg, 0.080 mg/kg, 0.085mg/kg, 0.090 mg/kg, 0.095 mg/kg, 0.100 mg/kg, 0.105 mg/kg, 0.110 mg/kg,0.115 mg/kg, 0.120 mg/kg, 0.125 mg/kg, 0.130 mg/kg, 0.135 mg/kg, 0.140mg/kg, 0.145 mg/kg, 0.150 mg/kg, 0.155 mg/kg, 0.160 mg/kg, 0.165 mg/kg,0.170 mg/kg, 0.175 mg/kg, 0.180 mg/kg, 0.185 mg/kg, 0.190 mg/kg, 0.195mg/kg, 0.200 mg/kg, 0.205 mg/kg, 0.210 mg/kg, 0.215 mg/kg, 0.220 mg/kg,0.225 mg/kg, 0.230 mg/kg, 0.235 mg/kg, 0.240 mg/kg, 0.245 mg/kg, 0.250mg/kg, 0.255 mg/kg, 0.260 mg/kg, 0.265 mg/kg, 0.270 mg/kg, 0.275 mg/kg,0.280 mg/kg, 0.285 mg/kg, 0.290 mg/kg, 0.295 mg/kg, 0.300 mg/kg, 0.305mg/kg, 0.310 mg/kg, 0.315 mg/kg, 0.320 mg/kg, 0.325 mg/kg, 0.330 mg/kg,0.335 mg/kg, 0.340 mg/kg, 0.345 mg/kg, 0.350 mg/kg, 0.355 mg/kg, 0.360mg/kg, 0.365 mg/kg, 0.370 mg/kg, 0.375 mg/kg, 0.380 mg/kg, 0.385 mg/kg,0.390 mg/kg, 0.395 mg/kg, 0.400 mg/kg, 0.405 mg/kg, 0.410 mg/kg, 0.415mg/kg, 0.420 mg/kg, 0.425 mg/kg, 0.430 mg/kg, 0.435 mg/kg, 0.440 mg/kg,0.445 mg/kg, 0.450 mg/kg, 0.455 mg/kg, 0.460 mg/kg, 0.465 mg/kg, 0.470mg/kg, 0.475 mg/kg, 0.480 mg/kg, 0.485 mg/kg, 0.490 mg/kg, 0.495 mg/kg,0.500 mg/kg immunosuppressant, such as rapamycin.

Exemplary doses of compositions comprising synthetic nanocarrierscomprising immunosuppressant, such as rapamycin, as provided herein canbe 0.55 mg/kg, 0.56 mg/kg, 0.57 mg/kg, 0.58 mg/kg, 0.59 mg/kg, 0.60mg/kg, 0.61 mg/kg, 0.62 mg/kg, 0.63 mg/kg,0.64 mg/kg, 0.65 mg/kg, 0.66mg/kg, 0.67 mg/kg, 0.68 mg/kg, 0.69 mg/kg, 0.70 mg/kg, 0.71 mg/kg, 0.72mg/kg, 0.73 mg/kg, 0.74 mg/kg, 0.75 mg/kg, 0.76 mg/kg, 0.77 mg/kg, 0.78mg/kg, 0.79 mg/kg, 0.80 mg/kg, 0.81 mg/kg, 0.82 mg/kg, 0.83 mg/kg, 0.84mg/kg, 0.85 mg/kg, 0.86 mg/kg, 0.87 mg/kg, 0.88 mg/kg, 0.89 mg/kg, 0.90mg/kg, 0.91 mg/kg, 0.92 mg/kg, 0.93 mg/kg, 0.94 mg/kg, 0.95 mg/kg, 0.96mg/kg, 0.97 mg/kg, 0.98 mg/kg, 0.90 mg/kg, 1.0 mg/kg, 1.01 mg/kg, 1.02mg/kg, 1.03 mg/kg, 1.04 mg/kg, 1.05 mg/kg, 1.06 mg/kg, 1.07 mg/kg, 1.08mg/kg, 1.09 mg/kg, 1.10 mg/kg, 1.11 mg/kg, 1.12 mg/kg, 1.13 mg/kg, 1.14mg/kg, 1.15 mg/kg, 1.16 mg/kg, 1.17 mg/kg, 1.18 mg/kg, 1.19 mg/kg, 1.20mg/kg, 1.21 mg/kg, 1.22 mg/kg, 1.23 mg/kg, 1.24 mg/kg, 1.25 mg/kg, 1.26mg/kg, 1.27 mg/kg, 1.28 mg/kg, 1.29 mg/kg, 1.30 mg/kg, 1.31 mg/kg, 1.32mg/kg, 1.33 mg/kg, 1.34 mg/kg, 1.35 mg/kg, 1.36 mg/kg, 1.37 mg/kg, 1.38mg/kg, 1.39 mg/kg, 1.40 mg/kg, 1.41 mg/kg, 1.42 mg/kg, 1.43 mg/kg, 1.44mg/kg, 1.45 mg/kg, 1.46 mg/kg, 1.47 mg/kg, 1.48 mg/kg, 1.49 mg/kg, 1.50mg/kg, 1.51 mg/kg, 1.52 mg/kg, 1.53 mg/kg, 1.54 mg/kg, 1.55 mg/kg, 1.56mg/kg, 1.57 mg/kg, 1.58 mg/kg, 1.59 mg/kg, 1.60 mg/kg, 1.61 mg/kg, 1.62mg/kg, 1.63 mg/kg, 1.64 mg/kg, 1.65 mg/kg, 1.66 mg/kg, 1.67 mg/kg, 1.68mg/kg, 1.69 mg/kg, 1.70 mg/kg, 1.71 mg/kg, 1.72 mg/kg, 1.73 mg/kg, 1.74mg/kg, 1.75 mg/kg, 1.76 mg/kg, 1.77 mg/kg, 1.78 mg/kg, 1.79 mg/kg, 1.80mg/kg, 1.81 mg/kg, 1.82 mg/kg, 1.83 mg/kg, 1.84 mg/kg, 1.85 mg/kg, 1.86mg/kg, 1.87 mg/kg, 1.88 mg/kg, 1.89 mg/kg, 1.90 mg/kg, 1.91 mg/kg, 1.92mg/kg, 1.93 mg/kg, 1.94 mg/kg, 1.95 mg/kg, 1.96 mg/kg, 1.97 mg/kg, 1.98mg/kg, 1.99 mg/kg, 2.00 mg/kg, 2.01 mg/kg, 2.02 mg/kg, 2.03 mg/kg, 2.04mg/kg, 2.05 mg/kg, 2.06 mg/kg, 2.07 mg/kg, 2.08 mg/kg, 2.09 mg/kg, 2.10mg/kg, 2.11 mg/kg, 2.12 mg/kg, 2.13 mg/kg, 2.14 mg/kg, 2.15 mg/kg, 2.16mg/kg, 2.17 mg/kg, 2.18 mg/kg, 2.19 mg/kg, 2.20 mg/kg, 2.21 mg/kg, 2.22mg/kg, 2.23 mg/kg, 2.24 mg/kg, 2.25 mg/kg, 2.26 mg/kg, 2.27 mg/kg, 2.28mg/kg, 2.29 mg/kg, 2.30 mg/kg, 2.31 mg/kg, 2.32 mg/kg, 2.33 mg/kg, 2.34mg/kg, 2.35 mg/kg, 2.36 mg/kg, 2.37 mg/kg, 2.38 mg/kg, 2.39 mg/kg, 2.40mg/kg, 2.41 mg/kg, 2.42 mg/kg, 2.43 mg/kg, 2.44 mg/kg, 2.45 mg/kg, 2.46mg/kg, 2.47 mg/kg, 2.48 mg/kg, 2.49 mg/kg, 2.50 mg/kg, 2.51 mg/kg, 2.52mg/kg, 2.53 mg/kg, 2.54 mg/kg, 2.55 mg/kg, 2.56 mg/kg, 2.57 mg/kg, 2.58mg/kg, 2.59 mg/kg, 2.60 mg/kg, 2.61 mg/kg, 2.62 mg/kg, 2.63 mg/kg, 2.64mg/kg, 2.65 mg/kg, 2.66 mg/kg, 2.67 mg/kg, 2.68 mg/kg, 2.69 mg/kg, 2.70mg/kg, 2.71 mg/kg, 2.72 mg/kg, 2.73 mg/kg, 2.74 mg/kg, 2.75 mg/kg, 2.76mg/kg, 2.77 mg/kg, 2.78 mg/kg, 2.79 mg/kg, 2.80 mg/kg, 2.81 mg/kg, 2.82mg/kg, 2.83 mg/kg, 2.84 mg/kg, 2.85 mg/kg, 2.86 mg/kg, 2.87 mg/kg, 2.88mg/kg, 2.89 mg/kg, 2.90 mg/kg, 2.91 mg/kg, 2.92 mg/kg, 2.93 mg/kg, 2.94mg/kg, 2.95 mg/kg, 2.96 mg/kg, 2.97 mg/kg, 2.98 mg/kg, 2.99 mg/kg, 3.00mg/kg, 3.01 mg/kg, 3.02 mg/kg, 3.03 mg/kg, 3.04 mg/kg, 3.05 mg/kg, 3.06mg/kg, 3.07 mg/kg, 3.08 mg/kg, 3.09 mg/kg, 3.10 mg/kg, 3.11 mg/kg, 3.12mg/kg, 3.13 mg/kg, 3.14 mg/kg, 3.15 mg/kg, 3.16 mg/kg, 3.17 mg/kg, 3.18mg/kg, 3.19 mg/kg, 3.20 mg/kg, 3.21 mg/kg, 3.22 mg/kg, 3.23 mg/kg, 3.24mg/kg, 3.25 mg/kg, 3.26 mg/kg, 3.27 mg/kg, 3.28 mg/kg, 3.29 mg/kg, 3.30mg/kg, 3.31 mg/kg, 3.32 mg/kg, 3.33 mg/kg, 3.34 mg/kg, 3.35 mg/kg, 3.36mg/kg, 3.37 mg/kg, 3.38 mg/kg, 3.39 mg/kg, 3.40 mg/kg, 3.41 mg/kg, 3.42mg/kg, 3.43 mg/kg, 3.44 mg/kg, 3.45 mg/kg, 3.46 mg/kg, 3.47 mg/kg, 3.48mg/kg, 3.49 mg/kg, 3.50 mg/kg, 3.51 mg/kg, 3.52 mg/kg, 3.53 mg/kg, 3.54mg/kg, 3.55 mg/kg, 3.56 mg/kg, 3.57 mg/kg, 3.58 mg/kg, 3.59 mg/kg, 3.60mg/kg, 3.61 mg/kg, 3.62 mg/kg, 3.63 mg/kg, 3.64 mg/kg, 3.65 mg/kg, 3.66mg/kg, 3.67 mg/kg, 3.68 mg/kg, 3.69 mg/kg, 3.70 mg/kg, 3.71 mg/kg, 3.72mg/kg, 3.73 mg/kg, 3.74 mg/kg, 3.75 mg/kg, 3.76 mg/kg, 3.77 mg/kg, 3.78mg/kg, 3.79 mg/kg, 3.80 mg/kg, 3.81 mg/kg, 3.82 mg/kg, 3.83 mg/kg, 3.84mg/kg, 3.85 mg/kg, 3.86 mg/kg, 3.87 mg/kg, 3.88 mg/kg, 3.89 mg/kg, 3.90mg/kg, 3.91 mg/kg, 3.92 mg/kg, 3.93 mg/kg, 3.94 mg/kg, 3.95 mg/kg, 3.96mg/kg, 3.97 mg/kg, 3.98 mg/kg, 3.99 mg/kg, 4.00 mg/kg, 4.01 mg/kg, 4.02mg/kg, 4.03 mg/kg, 4.04 mg/kg, 4.05 mg/kg, 4.06 mg/kg, 4.07 mg/kg, 4.08mg/kg, 4.09 mg/kg, 4.10 mg/kg, 4.11 mg/kg, 4.12 mg/kg, 4.13 mg/kg, 4.14mg/kg, 4.15 mg/kg, 4.16 mg/kg, 4.17 mg/kg, 4.18 mg/kg, 4.19 mg/kg, 4.20mg/kg, 4.21 mg/kg, 4.22 mg/kg, 4.23 mg/kg, 4.24 mg/kg, 4.25 mg/kg, 4.26mg/kg, 4.27 mg/kg, 4.28 mg/kg, 4.29 mg/kg, 4.30 mg/kg, 4.31 mg/kg, 4.32mg/kg, 4.33 mg/kg, 4.34 mg/kg, 4.35 mg/kg, 4.36 mg/kg, 4.37 mg/kg, 4.38mg/kg, 4.39 mg/kg, 4.40 mg/kg, 4.41 mg/kg, 4.42 mg/kg, 4.43 mg/kg, 4.44mg/kg, 4.45 mg/kg, 4.46 mg/kg, 4.47 mg/kg, 4.48 mg/kg, 4.49 mg/kg, 4.50mg/kg, 4.51 mg/kg, 4.52 mg/kg, 4.53 mg/kg, 4.54 mg/kg, 4.55 mg/kg, 4.56mg/kg, 4.57 mg/kg, 4.58 mg/kg, 4.59 mg/kg, 4.60 mg/kg, 4.61 mg/kg, 4.62mg/kg, 4.63 mg/kg, 4.64 mg/kg, 4.65 mg/kg, 4.66 mg/kg, 4.67 mg/kg, 4.68mg/kg, 4.69 mg/kg, 4.70 mg/kg, 4.71 mg/kg, 4.72 mg/kg, 4.73 mg/kg, 4.74mg/kg, 4.75 mg/kg, 4.76 mg/kg, 4.77 mg/kg, 4.78 mg/kg, 4.79 mg/kg, 4.80mg/kg, 4.81 mg/kg, 4.82 mg/kg, 4.83 mg/kg, 4.84 mg/kg, 4.85 mg/kg, 4.86mg/kg, 4.87 mg/kg, 4.88 mg/kg, 4.89 mg/kg, 4.90 mg/kg, 4.91 mg/kg, 4.92mg/kg, 4.93 mg/kg, 4.94 mg/kg, 4.95 mg/kg, 4.96 mg/kg, 4.97 mg/kg, 4.98mg/kg, 4.99 mg/kg, 5.00 mg/kg, 5.01 mg/kg, 5.02 mg/kg, 5.03 mg/kg, 5.04mg/kg, 5.05 mg/kg, 5.06 mg/kg, 5.07 mg/kg, 5.08 mg/kg, 5.09 mg/kg, 5.10mg/kg, 5.11 mg/kg, 5.12 mg/kg, 5.13 mg/kg, 5.14 mg/kg, 5.15 mg/kg, 5.16mg/kg, 5.17 mg/kg, 5.18 mg/kg, 5.19 mg/kg, 5.20 mg/kg, 5.21 mg/kg, 5.22mg/kg, 5.23 mg/kg, 5.24 mg/kg, 5.25 mg/kg, 5.26 mg/kg, 5.27 mg/kg, 5.28mg/kg, 5.29 mg/kg, 5.30 mg/kg, 5.31 mg/kg, 5.32 mg/kg, 5.33 mg/kg, 5.34mg/kg, 5.35 mg/kg, 5.36 mg/kg, 5.37 mg/kg, 5.38 mg/kg, 5.39 mg/kg, 5.40mg/kg, 5.41 mg/kg, 5.42 mg/kg, 5.43 mg/kg, 5.44 mg/kg, 5.45 mg/kg, 5.46mg/kg, 5.47 mg/kg, 5.48 mg/kg, 5.49 mg/kg, 5.50 mg/kg, 5.51 mg/kg, 5.52mg/kg, 5.53 mg/kg, 5.54 mg/kg, 5.55 mg/kg, 5.56 mg/kg, 5.57 mg/kg, 5.58mg/kg, 5.59 mg/kg, 5.60 mg/kg, 5.61 mg/kg, 5.62 mg/kg, 5.63 mg/kg, 5.64mg/kg, 5.65 mg/kg, 5.66 mg/kg, 5.67 mg/kg, 5.68 mg/kg, 5.69 mg/kg, 5.70mg/kg, 5.71 mg/kg, 5.72 mg/kg, 5.73 mg/kg, 5.74 mg/kg, 5.75 mg/kg, 5.76mg/kg, 5.77 mg/kg, 5.78 mg/kg, 5.79 mg/kg, 5.80 mg/kg, 5.81 mg/kg, 5.82mg/kg, 5.83 mg/kg, 5.84 mg/kg, 5.85 mg/kg, 5.86 mg/kg, 5.87 mg/kg, 5.88mg/kg, 5.89 mg/kg, 5.90 mg/kg, 5.91 mg/kg, 5.92 mg/kg, 5.93 mg/kg, 5.94mg/kg, 5.95 mg/kg, 5.96 mg/kg, 5.97 mg/kg, 5.98 mg/kg, 5.99 mg/kg, 6.00mg/kg, 6.01 mg/kg, 6.02 mg/kg, 6.03 mg/kg, 6.04 mg/kg, 6.05 mg/kg, 6.06mg/kg, 6.07 mg/kg, 6.08 mg/kg, 6.09 mg/kg, 6.10 mg/kg, 6.11 mg/kg, 6.12mg/kg, 6.13 mg/kg, 6.14 mg/kg, 6.15 mg/kg, 6.16 mg/kg, 6.17 mg/kg, 6.18mg/kg, 6.19 mg/kg, 6.20 mg/kg, 6.21 mg/kg, 6.22 mg/kg, 6.23 mg/kg, 6.24mg/kg, 6.25 mg/kg, 6.26 mg/kg, 6.27 mg/kg, 6.28 mg/kg, 6.29 mg/kg, 6.30mg/kg, 6.31 mg/kg, 6.32 mg/kg, 6.33 mg/kg, 6.34 mg/kg, 6.35 mg/kg, 6.36mg/kg, 6.37 mg/kg, 6.38 mg/kg, 6.39 mg/kg, 6.40 mg/kg, 6.41 mg/kg, 6.42mg/kg, 6.43 mg/kg, 6.44 mg/kg, 6.45 mg/kg, 6.46 mg/kg, 6.47 mg/kg, 6.48mg/kg, 6.49 mg/kg, or 6.50 mg/kg, for any one of the compositions ormethods provided, wherein the dose is given as the mg of the syntheticnanocarriers comprising the immunosuppressant, such as rapamycin.

Any one of the doses provided herein for the composition comprisinguricase, such as pegsiticase (i.e., pegadricase), can be used in any oneof the methods or compositions or kits provided herein. Any one of thedoses provided herein for the composition comprising syntheticnanocarriers comprising an immunosuppressant, such as rapamycin, can beused in any one of the methods or compositions or kits provided herein.Any one of the doses provided herein for the composition comprising ananti-inflammatory therapeutic can be used in any one of the methods orcompositions or kits provided herein. Any one of the doses providedherein for the composition(s) comprising an infusion reactiontherapeutic can be used in any one of the methods or compositions orkits provided herein. Generally, when referring to a dose to beadministered to a subject the dose is a label dose. Thus, in any one ofthe methods or composition provided herein the dose(s) are labeldose(s).

In some embodiments of any one of the methods provided herein, anadditional volume (prime volume) may be used to prime the infusion linefor administering any of the compositions provided herein to thesubject.

Provided herein are a number of possible dosing schedules. Accordingly,any one of the subjects provided herein may be treated according to anyone of the dosing schedules provided herein. As an example, any one ofthe subject provided herein may be treated with a composition comprisinguricase, such as pegylated uricase, and/or composition comprisingsynthetic nanocarriers comprising an immunosuppressant, such asrapamycin, and/or a composition comprising an anti-inflammatorytherapeutic and/or a composition(s) comprising an infusion reactiontherapeutic according to any one of these dosage schedules.

The mode of administration for the composition(s) of any one of thetreatment methods provided may be by intravenous administration, such asan intravenous infusion that, for example, may take place over about 1hour. Additionally, any one of the methods of treatment provided hereinmay also include administration of an additional therapeutic, such as auric acid lowering therapeutic, such as a uricase, or an anti-gout flareprophylactic treatment. The administration of the additional therapeuticmay be according to any one of the applicable treatment regimens and/ormodes of administration as provided herein.

Preferably, in some embodiments, the treatment with a combination ofsynthetic nanocarrier composition comprising immunosuppressant, such asrapamycin, with a composition comprising uricase, such as pegylateduricase, can comprise three doses of the synthetic nanocarriercomposition concomitantly with the uricase-comprising compositionfollowed by two doses of uricase without the concomitant administrationof a composition comprising an immunosuppressant, such as a syntheticnanocarrier composition comprising an immunosuppressant, with or withoutthe concomitant administration of an additional therapeutic. In such anembodiment, each dose may be administered every two to four weeks. Inone embodiment, a method is provided whereby any one of the subjectsprovided herein is concomitantly administered three doses of a syntheticnanocarrier composition with a uricase-comprising composition monthlyfor three months. In another embodiment, this method further comprisesadministering 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more monthly doses of auricase-comprising composition alone or without the concomitantadministration of immunosuppressant, such as a synthetic nanocarriercomposition comprising an immunosuppressant, or an additionaltherapeutic. In some embodiments of any one of the methods providedherein, the level of uric acid is measured in the subject at one or moretime points before, during and/or after the treatment period.

Subjects

Subjects provided herein can be in need of treatment according to anyone of the methods or compositions or kits provided herein. Suchsubjects include those with elevated serum uric acid levels or uric aciddeposits. Such subjects include those with hyperuricemia. It is withinthe skill of a clinician to be able to determine subjects in need of atreatment as provided herein.

In some embodiments, any one of the subjects for treatment as providedin any one of the methods provided has gout or a condition associatedwith gout or another condition as provided herein. In some embodiments,any one of the subjects for treatment as provided in any one of themethods provided the subject has had or is expected to have gout flare.

In some embodiments, the subject has or is at risk of having erosivebone disease associated with gout, cirrhosis or steathohepatitisassociated with gout, or visceral gout.

In some embodiments, the subject has or is at risk of having an elevateduric acid level, e.g., an elevated plasma or serum uric acid level. Whenblood levels of uric acid may exceed the physiologic limit ofsolubility, the uric acid may crystallize in the tissues, including thejoints, and may cause gout and gout-associated conditions.

In some embodiments, serum uric acid levels ≥ 5 mg/dL, ≥ 6 mg/dL, or ≥ 7mg/dL are indicative that a subject may be a candidate for treatmentwith any one of the methods or compositions or kits described herein. Insome embodiments, such a subject has a serum level of uric acid ≥ 6mg/dL, for example, between 6.1 mg/dL - 15 mg/dL, between 6.1 mg/dL - 10mg/dL, 7 mg/dL - 15 mg/dL, 7 mg/dL - 10 mg/dL, 8 mg/dL - 15 mg/dL, 8mg/dL - 10 mg/dL, 9 mg/dL -15 mg/dL, 9 mg/dL - 10 mg/dL, 10 mg/dL-15mg/dL, or 11 mg/dL- 14 mg/dL. In some embodiments, the subject has serumlevel of uric acid of about 6.1 mg/dL, 6.2 mg/dL, 6.3 mg/dL, 6.4 mg/dL,6.5 mg/dL, 6.7 mg/dL, 6.8 mg/dL, 6.9 mg/dL, 7.0 mg/dL, 7.1 mg/dL, 7.2mg/dL, 7.3 mg/dL, 7.4 mg/dL, 7.5 mg/dL,7.6 mg/dL 7.7 mg/dL, 7.8 mg/dL,7.9 mg/dL, 8.0 mg/dL, 8.1 mg/dL, 8.2 mg/dL, 8.3 mg/dL, 8.4 mg/dL, 8.5mg/dL, 8.6 mg/dL, 8.7 mg/dL, 8.8 mg/dL, 8.9 mg/dL, 9.0 mg/dL, 9.1 mg/dL,9.2 mg/dL, 9.3 mg/dL, 9.4 mg/dL, 9.5 mg/dL, 9.6 mg/dL, 9.7 mg/dL, 9.8mg/dL, 9.9 mg/dL, 10.0 mg/dL, 10.1 mg/dL, 10.2 mg/dL, 10.3 mg/dL, 10.4mg/dL, 10.5 mg/dL, 10.6 mg/dL, 10.7 mg/dL, 10.8 mg/dL, 10.9 mg/dL, 11.0mg/dL, 11.1 mg/dL, 11.2 mg/dL, 11.3 mg/dL, 11.4 mg/dL, 11.5 mg/dL, 11.6mg/dL, 11.7 mg/dL, 11.8 mg/dL, 11.9 mg/dL, 12.0 mg/dL, 12.1 mg/dL, 12.2mg/dL, 12.3 mg/dL, 12.4 mg/dL, 12.5 mg/dL, 12.6 mg/dL, 12.7 mg/dL, 12.8mg/dL, 12.9 mg/dL, 13.0 mg/dL, 13.1 mg/dL, 13.2 mg/dL, 13.3 mg/dL, 13.4mg/dL, 13.5 mg/dL, 13.6 mg/dL, 13.7 mg/dL, 13.8 mg/dL, 13.9 mg/dL, 14.0mg/dL, 14.1 mg/dL, 14.2 mg/dL, 14.3 mg/dL, 14.4 mg/dL, 14.5 mg/dL, 14.6mg/dL, 14.7 mg/dL, 14.8 mg/dL, 14.9 mg/dL, 15.0 mg/dL or higher. In someembodiments, the subject has a plasma or serum uric acid level of 5.0mg/dL, 5.1 mg/dL, 5.2 mg/dL, 5.3 mg/dL, 5.4 mg/dL, 5.5 mg/dL, 5.6 mg/dL,5.7 mg/dL, 5.8 mg/dL, 5.9 mg/dL, 6.0 mg/dL, 6.1 mg/dL, 6.2 mg/dL, 6.3mg/dL, 6.4 mg/dL, 6.5 mg/dL, 6.6 mg/dL, 6.7 mg/dL, 6.8 mg/dL, 6.9 mg/dL,or 7.0 mg/dL. In some embodiments, the subject has a plasma or serumuric acid level of greater than or equal to 5.0 mg/dL, 5.1 mg/dL, 5.2mg/dL, 5.3 mg/dL, 5.4 mg/dL, 5.5 mg/dL, 5.6 mg/dL, 5.7 mg/dL, 5.8 mg/dL,5.9 mg/dL, 6.0 mg/dL, 6.1 mg/dL, 6.2 mg/dL, 6.3 mg/dL, 6.4 mg/dL, 6.5mg/dL, 6.6 mg/dL, 6.7 mg/dL, 6.8 mg/dL, 6.9 mg/dL, or 7.0 mg/dL.

In some embodiments, the subject has, or is at risk of having,hyperuricemia. In some embodiments, the subject has, or is at risk ofhaving, gout, acute gout, acute intermittent gout, gouty arthritis,acute gouty arthritis, acute gouty arthropathy, acute polyarticulargout, recurrent gouty arthritis, chronic gout (with our without tophi),tophaceous gout, chronic tophaceous gout, chronic advanced gout (withour without tophi), chronic polyarticular gout (with our without tophi),chronic gouty arthropathy (with our without tophi), idiopathic gout,idiopathic chronic gout (with or without tophi), primary gout, chronicprimary gout (with or without tophi), refractory gout, such as chronicrefractory gout, axial gouty arthropathy, a gout attack, a gout flare,podagra (i.e., monarticular arthritis of the great toe), chiragra (i.e.,monarticular arthritis of the hand), gonagra (i.e., monarticulararthritis of the knee), gouty bursitis, gouty spondylitis, goutysynovitis, gouty tenosynovitis, gout that affects tendons and ligaments,lead-induced gout (i.e., saturnine gout), drug induced gout, gout due torenal impairment, gout due to kidney disease, chronic gout due to renalimpairment (with or without tophi), chronic gout due to kidney disease(with or without tophi), erosive bone disease associated with gout,stroke associated with gout, vascular plaque associated with gout,cirrhosis or steatohepatitis associated with gout, liver-associatedgout, incident and recurrent gout, diabetes associated with damage topancreas in gout, general inflammatory diseases exacerbated by gout,other secondary gout, or unspecified gout.

In some embodiments, the subject has, or is at risk of having, acondition associated with the renal system, for example, calculus ofurinary tract due to gout, uric acid urolithiasis, uric acidnephrolithiasis, uric acid kidney stones, gouty nephropathy, acute goutynephropathy, chronic gouty nephropathy, urate nephropathy, uric acidnephropathy, and gouty interstitial nephropathy.

In some embodiments, the subject has, or is at risk of having, acondition associated with the nervous system, for example, peripheralautonomic neuropathy due to gout, gouty neuropathy, gouty peripheralneuropathy, gouty entrapment neuropathy, or gouty neuritis.

In some embodiments, the subject has, or is at risk of having, acondition associated with the cardiovascular system, for example,metabolic syndrome, hypertension, obesity, diabetes, myocardialinfarction, stroke, dyslipidemia, hypertriglyceridemia, insulinresistance/hyperglycemia, coronary artery disease/coronary heartdisease, coronary artery disease or blockage associated with gout orhyperuricemia, heart failure, peripheral arterial disease,stroke/cerebrovascular disease, peripheral vascular disease, andcardiomyopathy due to gout.

In some embodiments, the subject has, or is at risk of having, acondition associated with the ocular system including, for example,gouty iritis, inflammatory disease in the eye caused by gout, dry eyesyndrome, red eye, uveitis, intraocular hypertension, glaucoma, andcataracts.

In some embodiments, the subject has, or is at risk of having, acondition associated with the skin including, for example, gout of theexternal ear, gouty dermatitis, gouty eczema, gouty panniculitis, andmiliarial gout.

Compositions and Kits

Compositions provided herein may comprise inorganic or organic buffers(e.g., sodium or potassium salts of phosphate, carbonate, acetate, orcitrate) and pH adjustment agents (e.g., hydrochloric acid, sodium orpotassium hydroxide, salts of citrate or acetate, amino acids and theirsalts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants(e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol,sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g.,sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g.,salts or sugars), antibacterial agents (e.g., benzoic acid, phenol,gentamicin), antifoaming agents (e.g., polydimethylsilozone),preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymericstabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone,poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol,polyethylene glycol, ethanol).

Compositions according to the invention may comprise pharmaceuticallyacceptable excipients. The compositions may be made using conventionalpharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. Techniques suitable for use in practicing thepresent invention may be found in Handbook of Industrial Mixing: Scienceand Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, andSuzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: TheScience of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001,Churchill Livingstone. In an embodiment, compositions are suspended in asterile saline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method of manufacture may require attentionto the properties of the particular elements being associated.

In some embodiments, compositions are manufactured under sterileconditions or are initially or terminally sterilized. This can ensurethat resulting compositions are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingthe compositions have immune defects, are suffering from infection,and/or are susceptible to infection. In some embodiments, thecompositions may be lyophilized and stored in suspension or aslyophilized powder depending on the formulation strategy for extendedperiods without losing activity.

Administration according to the present invention may be by a variety ofroutes, including but not limited to an intravenous route. Thecompositions referred to herein may be manufactured and prepared foradministration using conventional methods.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Dosesof compositions as provided herein may contain varying amounts ofelements according to the invention. The amount of elements present inthe compositions for dosing can be varied according to their nature, thetherapeutic benefit to be accomplished, and other such parameters. Thecompositions for dosing may be administered according to any one of thefrequencies provided herein.

Another aspect of the disclosure relates to kits. In some embodiments,the kit comprises any one or more of the compositions provided herein.In some embodiments of any one of the kits provided, the kit comprisesany one or more of the compositions comprising one or more compositionscomprising an anti-inflammatory therapeutic and/or an infusion reactiontherapeutic and one or more synthetic nanocarrier compositions providedherein. Any one or more of the kits can further comprise one or morecompositions comprising an uricase. Each of the types of compositionscan be in one container or in more than one container in the kit. Insome embodiments of any one of the kits provided, the container is avial or an ampoule. In some embodiments of any one of the kits provided,the composition(s) are in lyophilized form each in a separate containeror in the same container, such that they may be reconstituted at asubsequent time. In some embodiments of any one of the kits provided,the composition(s) are in the form of a frozen suspension each in aseparate container or in the same container, such that they may bereconstituted at a subsequent time. In some embodiments of any one ofthe kits, the frozen suspension further comprises PBS. In someembodiments of any one of the kits, the kit further comprises PBS and/or0.9% sodium chloride, USP.

In some embodiments of any one of the kits provided, the kit furthercomprises instructions for reconstitution, mixing, administration, etc.In some embodiments of any one of the kits provided, the instructionsinclude a description of any one of the methods described herein.Instructions can be in any suitable form, e.g., as a printed insert or alabel. In some embodiments of any one of the kits provided herein, thekit further comprises one or more syringes or other device(s) that candeliver the composition(s) in vivo to a subject.

EXAMPLES Example 1: SEL-212 Clinical Trial Results, Human Phase 1aClinical Trial

The Phase 1a clinical trial for SEL-212 was an ascending dose trial ofpegsiticase alone in 22 subjects with elevated serum uric acid levelsgreater than 6 mg/dl who were separated into five cohorts. Each cohortreceived a single intravenous infusion of pegsiticase at the followingdose levels of 0.1 mg/kg for Cohort #1, 0.2 mg/kg for Cohort #2, 0.4mg/kg for Cohort #3, 0.8 mg/kg for Cohort #4 and 1.2 mg/kg for Cohort#5. Dosing began with the lowest dose and only after an entire cohortwas safely dosed was the next cohort started. The subjects weremonitored during a 30 day period post infusion with visits occurring onday 7, 14, 21 and the end of trial visit on day 30. Blood and serum ofeach patient was evaluated for serum uric acid, ADAs (specificallyanti-peg, anti-uricase and anti-pegsiticase) and safety parameters. Itwas observed that pegsiticase demonstrated no serious adverse events andwas well tolerated at the five dose levels tested. Additionally, it wasobserved that pegsiticase rapidly reduced (within hours) and sustainedaverage serum uric acid levels below 6 mg/dl for each cohort for 14 to30 days, depending on the dose level. Consistent with preclinicalstudies in animals, pegsiticase induced uricase specific ADAs in allsubjects with varying levels in this Phase 1a trial.

FIG. 3 depicts average serum uric acid levels of the Phase 1a clinicaltrial’s five cohorts tested at different measurement intervals (Day 7,14, 21 and 30) during the course of the 30 day period following thesingle intravenous infusion of pegsiticase at the outset of the trial.

The serum uric acid levels were measured at baseline and days seven, 14,21 and 30 and uricase specific ADA levels at baseline and days seven, 14and 30 following a single intravenous injection of pegsiticase. Uricasespecific ADA levels at day 21 in the Phase 1a clinical trial were notmeasured. Based on the results from the Phase 1a clinical trial, it wasobserved that pegsiticase at a tolerated dose is capable of achievingand maintaining a reduction of serum uric acid below the target of 6mg/dl for a 30 day period in the absence of inhibitory uricase specificADAs.

Phase 1b Clinical Trial

The Phase 1b clinical trial enrolled 63 patients with serum uric acidlevels greater than 6 mg/dl were separated into 11 cohorts. A singleintravenous infusion of SVP Rapamycin alone at the following ascendingdose levels was administered to four cohorts in ascending order. Eachcohort consisted of seven patients and they were designated as follows:Cohort #1 (0.03 mg/kg), Cohort #3 (0.1 mg/kg), Cohort #5 (0.3 mg/kg) andCohort #7 (0.5 mg/kg) collectively the SVP Rapamycin Cohorts. After acohort of the SVP-Rapamycin alone had successfully and safely been dosedthe corresponding dose level of SVP Rapamycin was combined with a fixeddose of pegsiticase (0.4 mg/kg). The combination was co-administeredsequentially as a single intravenous infusion, with the SVP Rapamycininfusion preceding the pegsiticase infusion. The cohort designation isas follows for the six cohorts (5 patients per cohort), which wereCohort #2 (SVP Rapamycin 0.03 mg/kg + 0.4 mg/kg pegsiticase), Cohort #4(SVP Rapamycin 0.1 mg/kg + 0.4 mg/kg pegsiticase), Cohort #6 (SVPRapamycin 0.3 mg/kg + 0.4 mg/kg pegsiticase), Cohort #10 (0.4 mg/kgpegsiticase + 0.03 mg/kg SVP Rapamycin separated by 48 hours), Cohort#12 (SVP Rapamycin 0.15 mg/kg + 0.4 mg/kg pegsiticase) and Cohort #14(SVP Rapamycin 0.1 mg/kg + 0.4 mg/kg pegsiticase) collectively theSEL-212 Cohorts. In Cohort #9 a fixed amount of pegsiticase alone at adose level of 0.4 mg/kg was administered to five patients, which isreferred to as the Pegsiticase Cohort. Methods of such treatment arealso provided. The subjects were monitored during a 30 day period postinfusion with visits occurring on day 7, 14, 21 and the end of trialvisit on day 30. Blood and serum of each patient was evaluated for serumuric acid, ADAs (specifically anti-PEG, anti-uricase andanti-pegsiticase) and safety parameters. The primary objective of thePhase 1b clinical trial was to evaluate the safety and tolerability ofSVP Rapamycin alone and in combination with a fixed dose of pegsiticase.A secondary clinical objective was to evaluate the ability of SVPRapamycin co-administered with pegsiticase to reduce serum uric acidlevels and mitigate the formation of uricase specific ADAs when comparedto administration of pegsiticase alone.

FIG. 4 indicates the serum uric acid levels of Cohort #3 from the Phase1a clinical trial, in which subjects received a fixed amount ofpegsiticase alone (at the same 0.4 mg/kg pegsiticase. Also in the firstgraph is the data from Cohort #9 (pegsiticase 0.4 mg/kg) of the Phase 1bclinical trial. This graph represents the reproducibility of the dataacross two separate studies. In both cohorts there is initial control ofthe serum uric acid (levels maintained below 6 mg/dL) but past day 14,individuals loose the enzyme activity. Also in FIG. 4 , the data fromthe SVP rapamycin alone cohort is displayed. All values remainessentially the same throughout the 30 days of testing indicating thatSVP Rapamycin alone has no effect on serum uric acid levels. For Cohort#2 from the Phase 1b clinical trial, which received the lowest dose ofSVP Rapamycin co-administered with pegsiticase, it was observed thatfour out of five subjects tested maintained serum uric acid levels below6 mg/dl through day 21 of the trial. It was also observed that four outof five subjects in Cohort #4 from the Phase 1b clinical trial, whichreceived the second lowest dose of SVP Rapamycin co-administered withpegsiticase, maintained levels of serum uric acid of less than 0.1 mg/dlthrough day 30. For Cohort #6 (SEL-212 Cohort), it was observed thatfour (out of the projected five) subjects maintained levels of serumuric acid of less than 0.1 mg/dl through day 21 and two (out of theprojected five) subjects maintained levels of serum uric acid of lessthan 0.1 mg/dl through day 30. By comparison, for Cohort #9 (PegsiticaseCohort), four of the five subjects returned to baseline serum uric acidlevels by day 30.

FIG. 4 shows the serum uric acid levels and uricase specific ADA levelsfor each subject in Cohort #3 of the Phase 1a clinical trial and Cohort#9 (Pegsiticase Cohort) of the Phase 1b clinical trial for comparison tothe serum uric acid levels and uricase specific ADA levels for eachsubject in Cohort # 4 (SEL-212 Cohort) in the Phase 1b clinical trial.Cohort #3 from the Phase 1a clinical trial is depicted along with Cohort#9 from the Phase 1b clinical trial for purposes of comparison againstCohort #4 from the Phase 1b clinical trial because the subjects in thesecohorts received the same fixed dose of pegsiticase. In addition, Cohort#4 from the Phase 1b clinical trial is depicted in FIG. 4 because thesubjects in Cohort #4 from the Phase 1b clinical trial received a higherdose of SVP Rapamycin than did the subjects in Cohort #2 in the Phase 1bclinical trial, the other SEL-212 Cohort for which 30 day observationperiod data from the Phase 1b clinical trial was available.

As depicted in FIG. 4 , in Cohort #3 from the Phase 1a clinical trialand Cohort #9 from the Phase 1b clinical trial, uricase specific ADAformation at day 14 resulting in a return to baseline levels of serumuric acid was observed. In comparison, for Cohort #4 from the Phase 1bclinical trial, it was observed that minimal uricase specific ADAformation in four of the five subjects tested with correspondingmaintenance of control of serum uric acid levels through day 30. In thePhase 1a clinical trial, uricase specific ADA levels at day 21 was notmeasured. However, in the course of conducting the Phase 1a clinicaltrial, it was learned that it would be useful to measure uricasespecific ADA levels at day 21 to more fully understand any variations insuch levels between day 14 and day 30. As a result, for the Phase 1bclinical trial, uricase specific ADA levels at day 21 were monitored.

Additional serum uric acid and uricase specific ADA data after day 30was collected for three of the subjects in Cohort #4 (SEL-212 Cohort)that had no or very low serum uric acid and uricase specific ADA levelsat day 30. Data on day 37 was collected for all three of these subjectsand again on day 42 or day 44 for two of the three subjects. Each ofthese three subjects had no or very low uricase specific ADA levels onday 37, day 42 or day 44, as applicable. Serum uric acid levels remainedbelow baseline on day 37 in all three subjects. With respect to the twosubjects for which day 42 or day 44 data was available, serum uric acidlevels approached or exceeded baseline by the last time point measured.Based on the observations from the Phase 1b clinical trial data, it wasfound that SEL-212 was capable of controlling uric acid levels for atleast 30 days in the majority of subjects in Cohort # 4.

On a combined basis, a total of 85 subjects have been dosed with eitherSEL-212 (SVP Rapamycin and pegsiticase), SVP Rapamycin alone orpegsiticase alone in connection with the Phase 1a and Phase 1b clinicaltrials. It has been generally observed that SEL-212 and its components,SVP Rapamycin and pegsiticase, have been well tolerated. There have beena total of four serious adverse events, or SAEs, in both Phase 1clinical trials. All SAEs fully resolved.

FIG. 5 shows the serum uric acid levels and uricase-specific ADA levelsfor each subject in Cohort #3 of the Phase 1a clinical trial and Cohort#9 (Pegsiticase Cohort) of the Phase 1b clinical trial for comparison tothe serum uric acid levels and uricase-specific ADA levels for eachsubject in Cohort # 4 (SEL-212 Cohort) and Cohort #6 (SEL-212 Cohort) inthe Phase 1b clinical trial. Cohort #3 from the Phase 1a clinical trialis also depicted along with Cohort #9 from the Phase 1b clinical trialfor purposes of comparison against Cohort #4 and Cohort #6 from thePhase 1b clinical trial because the subjects in these cohorts receivedthe same fixed dose of pegsiticase. In addition, Cohort #4 from thePhase 1b clinical trial is depicted because the subjects in Cohort #4from the Phase 1b clinical trial received a higher dose of SVP-Rapamycinthan did the subjects in Cohort #2 in the Phase 1b clinical trial. Alsoincluded is Cohort #6 from the Phase 1b clinical trial because thesesubjects received the highest dose of SVP-Rapamycin tested todate-higher than both Cohorts #2 and #4.

FIG. 6 presents a non-head-to-head comparison of the efficacy of SEL-212in Cohort #6 of the Phase 1b clinical trial with Cohort #5 of the Phase1b clinical trial and data from two replicate, randomized, double-blind,placebo-controlled clinical trials of KRYSTEXXA® as reported in theJournal of the American Medical Association in 2011. These twoKRYSTEXXA® clinical trials included 85 patients who received biweeklydoses of KRYSTEXXA®, 84 patients who received monthly doses ofKRYSTEXXA® and 43 patients who received a placebo.

KRYSTEXXA® has been approved for the treatment of refractory gout on abiweekly dose regimen whereas the monthly dose regimen of KRYSTEXXA® hasnot been approved for marketing. The graph on the left below depicts thedata for the four-week period after the first dose of Krystexxa® fromthe cohorts of subjects in the KRYSTEXXA® clinical trials who receivedmonthly doses.

The placebo control subjects, indicated in open circles in FIG. 6 , haduric acid levels above 6 mg/dl for the entire four weeks. TheKRYSTEXXA®-treated subjects that went on to become responders, asdefined by maintenance of uric acid levels below 6 mg/dl for 80% of thetime at months three and six, are indicated in black circles. TheKRYSTEXXA®treated subjects that went on to become non-responders, asdefined by the inability to maintain uric acid levels below 6 mg/dl for80% of the time at months three and six, are indicated in blacktriangles. Only 35% of KRYSTEXXA®-treated subjects in the monthly dosingcohorts were classified as responders. It is notable that, even at fourweeks, the mean uric acid levels were above 6 mg/dl in thenon-responders, representing 65% of subjects, and were above 4 mg/dl inthe responders. 89% of all KRYSTEXXA®-treated subjects developed ADAs.In comparison, the graph on the right in FIG. 6 depicts data from Cohort#5 of the Phase 1b clinical trial, which received a single dose ofSVP-Rapamycin alone, and Cohort #6 of the Phase 1b clinical trial, whichreceived a single dose of SEL-212. All five subjects in Cohort #6 of thePhase 1b clinical trial, treated with SEL-212, maintained levels ofserum uric acid of less than 0.1 mg/dl through day 30. Subjects inCohort #5 of the Phase 1b clinical trial, treated with SVP-Rapamycinalone, experienced no significant reduction in uric acid levels, as suchlevels remained relatively constant over the 30-day period. Also shownis a comparison of data from Cohort #5 of the Phase 1b clinical trial,which received a single dose of SVP-Rapamycin alone, with Cohort #9 ofthe Phase 1b clinical trial, which received pegstiticase alone.

While it is believed that the above comparison is useful in evaluatingthe results of Cohort #6 of the Phase 1b clinical trial, the Phase 1bclinical trial and the KRYSTEXXA® clinical trials were separate trialsconducted by different investigators at different sites. In addition,there were substantial differences, including, for example, that theKRYSTEXXA® clinical trials were double-blind trials involving asubstantial number of patients with refractory gout while the Phase 1bclinical trial evaluated SEL-212 in an unblended manner in a smallnumber of subjects with elevated uric acid levels. Moreover, only theefficacy of SEL-212 with the four-week period following the firstinjection of KRYSTEXXA® could be compared as SEL-212 had not yet beenevaluated in a multi-dose clinical trial.

Additional serum uric acid and uricase-specific ADA data was collectedafter day 30 for three of the subjects in Cohort #4 (SEL-212 Cohort)that had no or very low serum uric acid and uricase-specific ADA levelsat day 30. Data was collected on day 37 for all three of these subjectsand again on day 42 or day 44 for two of the three subjects. Each ofthese three subjects had no or very low uricase-specific ADA levels onday 37, day 42 or day 44, as applicable. Serum uric acid levels remainedbelow baseline on day 37 in all three subjects. With respect to the twosubjects for which day 42 or day 44 data was available, serum uric acidlevels approached or exceeded baseline by the last time point measured.

Example 2 - Phase 2 Clinical Trial

Presented herein is a phase 2 clinical trial of SEL-212. The studyconsists of multiple doses of SEL-212 concomitantly administered withdoses of SEL-037. SEL-212 is a combination of SEL-037 and SEL-110.SEL-037 comprises pegsiticase (Recombinant Pegylated Candida UrateOxidase). SEL-110 is a nanocarrier comprising PLA (poly(D,L-lactide))and PLA-PEG (poly(D,L- lactide)-block-poly (ethylene-glycol))encapsulating rapamycin.

SEL-037 can be provided with phosphate buffer and mannitol asexcipients. Prior to administration, 6 mg, measured as uricase protein,lyophilized SEL-037 can be reconstituted with 1.1 ml of sterile waterfor injection, USP (United States Pharmacopeia) which forms a 6 mg/mLconcentrated solution. A sufficient volume of reconstituted SEL-037 at0.2 mg/kg or 0.4 mg/kg, measured as uricase protein, is diluted in 100mL of 0.9% sodium chloride for injection, USP and dosed as a singleintravenous infusion with an infusion pump over 60 minutes.

SEL-110 is provided as a 2 mg/mL, based on rapamycin content, suspensionin PBS. The appropriate amount of SEL-110 on a mg/kg basis is drawn intoa syringe or syringes and administered as an IV infusion with a syringeinfusion pump. If a subject is part of Cohorts 3, 4, 5, 6, 7 and 8 thenSEL-110 is administered prior to SEL-037. SEL-110 is delivered bysyringe infusion pump at a single steady rate sufficient to deliver thedose volume over a period of 55 minutes concurrently with a 60 minuteinfusion of 125 mL of normal saline and then the SEL-037 infusion (0.2mg/kg for Cohorts 3, 5 and 7; 0.4 mg/kg for Cohorts 4, 6 and 8) arestarted at the 60 minute mark.

96 subjects were divided into 11 dosing cohorts. Cohort 1 receives SEL037 (pegsiticase alone, 0.2 mg/kg), Cohort 2 receives SEL-037(pegsiticase alone, 0.4 mg/kg), Cohort 3 receives SEL-212 (with 0.05mg/kg of SEL-110 + 0.2 mg/kg pegsiticase), Cohort 4 receives SEL-212(with 0.05 mg/kg of SEL-110 + 0.4 mg/kg pegsiticase), Cohort 5 receivesSEL-212 (with 0.08 mg/kg of SEL-110 + 0.2 mg/kg pegsiticase), Cohort 6receives SEL-212 (with 0.08 mg/kg of SEL-110 + 0.4 mg/kg pegsiticase),Cohort 7 receives SEL-212 (with 0.1 mg/kg of SEL-110 + 0.2 mg/kgpegsiticase), Cohort 8 receives SEL-212 (with 0.1 mg/kg of SEL-110 + 0.4mg/kg pegsiticase), Cohort 10 receives SEL-212 (with 0.125 mg/kg ofSEL-110 + 0.4 mg/kg pegsiticase, Cohort 11 receives SEL-212 (with 0.15mg/kg of SEL-110 + 0.2 mg/kg pegsiticase), and Cohort 12 receivesSEL-212 (with 0.15 mg/kg of SEL-110 + 0.4 mg/kg pegsiticase.

Distribution of Subjects

All enrolled subjects were randomized initially to 4 cohorts such thatupon reaching 12 subjects total for all 4 cohorts, each cohort contains3 subjects. After the completion of at least one treatment cycle thesubject experience is evaluated before enrollment is opened to allcohorts. The future enrollment is randomized between all open cohorts.

Premedication for Study Drug Treatments

All subjects received 180 mg fexofenadine orally the night beforereceiving study drug (12 h ± 2 h) and again 2 ± 1 hours before receivingstudy drug (i.e., prior to SEL-037 for Cohorts 1 and 2 or SEL-110 forCohorts 3-8, 10, 11, and 12). In addition, they also receivedmethylprednisolone 40 mg (or equivalent drug, for example prednisone 50mg IV or dexamethasone 8 mg IV) intravenously 1 ± 0.5 hour beforereceiving study drug (i.e., prior to SEL-037 for Cohorts 1 and 2 orSEL-110 for Cohorts 3-8, 10, 11, and 12). This occurs for everytreatment dosing of study drug (Part A, Treatment Periods 1-3 and forPart B, Treatment Periods 4 and 5).

Premedication for Gout Flare

All subjects that met all inclusion and exclusion criteria were givenpremedication for gout flare prevention. The regimen began 1 week priorto the first dosing of study drug and continued for as long as thesubject was enrolled in the clinical study. Subjects were givencolchicine 1.2 mg as a single loading dose. Then they continued withcolchicine 0.6 mg QD for the remainder of their participation in thetrial. If there was a contraindication to colchicine, the subjectreceived ibuprofen 600 mg TID or equivalent dose of a NSAID. If there isa contraindication to colchicine and to NSAIDs the subject received nopremedication for gout flare. The gout flare prevention medicationcontinued as long as the subject was enrolled in the clinical study.Subjects who began receiving a NSAID as gout flare prevention medicationdue to a contraindication to colchicine continued to receive the NSAIDas long as the subject is enrolled in the study.

Duration of Treatment for Cohort 3, Cohort 4, Cohort 5, Cohort 6, Cohort7, Cohort 8, Cohort 10, Cohort 11, and Cohort 12 Treatment Period 1 -Part A

Subjects were screened within 45 days of dosing. Once they metinclusion/exclusion criteria and all assessments were consideredacceptable they were instructed on when to start their premedication(date and medication, Day -7) for the prevention of gout flares. The dayof initial dosing of study drug was designated Day 0. Eligible subjectswho have been assigned to Cohorts 3, 4, 5, 6, 7 and 8 received a singleIV in fusion of SEL-110 (dose based on a mg/kg basis). SEL-110 wasdelivered by syringe infusion pump at a single steady rate sufficient todeliver the dose volume over a period of 55 minutes. Concurrently to theadministration of SEL-110, the subject received a 125 mL of normalsaline over 60 minutes. This was followed (± 3 minutes) by an infusiondelivered by infusion pump of SEL-037 (0.2 mg/kg for Cohorts 3, 5, and7; 0.4 mg/kg for Cohorts 4, 6 and 8) diluted into 100 mL of normalsaline delivered over 60 minutes. Subjects remained in the clinic for 9hours after the start of the infusion of SEL-110 for safety evaluationsand PK blood draws. Subjects returned for PK and PD blood draws onTreatment Period 1, Days 1, 7, 14, 21 and safety andAntibody blood drawson Treatment Period 1, Days 7, 14, 21.

Treatment Period 2 - Part A

On the morning of Treatment Period 2, Day 0, subjects reported to theclinic for the dosing of study drug. Eligible subjects who had beenassigned to Cohorts 3, 4, 5, 6, 7 and 8 received a single IV infusion ofSEL-110 (dose based on a mg/kg basis). SEL-110 was delivered by syringeinfusion pump at a single steady rate sufficient to deliver the dosevolume over a period of 55 minutes. Concurrently to the administrationof SEL-110, the subject received a 125 mL of normal saline over 60minutes. This was followed (± 3 minutes) by an infusion delivered byinfusion pump of SEL-037 (0.2 mg/kg for Cohorts 3, 5 and 7; 0.4 mg/kgfor Cohorts 4, 6 and 8) diluted into 100 mL of normal saline deliveredover 60 minutes. Subjects remained in the clinic for 9 hours after thestart of the infusion of SEL-110 for safety evaluations and PK blooddraws. Subjects returned for PK and PD on Treatment Period 2, Days 1, 7,14 and 21 and safety and antibody blood draws on Treatment Period 2,Days 7, 14 and 21.

Treatment Period 3 - Part A

On the morning of Treatment Period 3, Day 0 subjects will report to theclinic for the dosing of study drug. Eligible subjects who have beenassigned to Cohorts 3, 4, 5, 6, 7 and 8 will receive a single IVinfusion of SEL-110 (dose based on a mg/kg basis). SEL-110 will bedelivered by syringe infusion pump at a single steady rate sufficient todeliver the dose volume over a period of 55 minutes. Concurrently to theadministration of SEL-110, the subject will receive a 125 mL of normalsaline over 60 minutes. This will be followed (± 3 minutes) by aninfusion delivered by infusion pump of SEL-037 (0.2 mg/kg for Cohorts 3,5 and 7; 0.4 mg/kg for Cohorts 4, 6 and 8) diluted into 100 mL of normalsaline delivered over 60 minutes. Subjects will remain in the clinic for9 hours after the start of the infusion of SEL-110 for safetyevaluations and PK blood draws. Subjects will return for PK and PD blooddraws on Treatment Period 3, Days 1, 7, 14 and 21 and safety andantibody blood draws on Treatment Period 3, Days 7, 14 and 21.

Treatment Period 4 - Part B

On the morning of Treatment Period 4, Day 0 subjects will report to theclinic for the dosing of study drug. Subjects will receive a single IVinfusion of SEL-037 (0.2 mg/kg for Cohorts 3, 5 and 7; 0.4 mg/kg forCohorts 4, 6 and 8) diluted into 100 mL of normal saline over 60 minutesby infusion pump. Subjects will remain in the clinic for 9 hours afterthe start of the infusion of SEL-037 for safety evaluations and PK blooddraws. Subjects will return for PK and PD blood draws on TreatmentPeriod 4, Days 1, 7, 14 and 21 and safety and antibody blood draws onTreatment Period 4, Days 7, 14 and 21.

Treatment Period 5 - Part B

On the morning of Treatment Period 5, Day 0 subjects will report to theclinic for the dosing of study drug. Subjects will receive a single IVinfusion of SEL-037 (0.2 mg/kg for Cohorts 3, 5 and 7; 0.4 mg/kg forCohorts 4, 6 and 8) diluted into 100 ml of normal saline over 60 minutesby infusion pump. Subjects will remain in the clinic for 9 hours afterthe start of the infusion of SEL-037 for safety evaluations and PK blooddraws. Subjects will return for PK and PD blood draws on TreatmentPeriod 5, Days 1, 7, 14 and 21 and safety and antibody blood draws onTreatment Period 5, Days 7, 14 and 21.

Results

When pegsiticase was administered alone in the Phase 1 described inExample 1, 24% (23 out of 96 patients) of those with a history of gouthad signs of gout flare in the first month after receiving the studydrug (Table 1). In contrast, however, when PLA/PLA-PEG syntheticnanocarriers comprising rapamycin were concomitantly administered withpegsiticase in a Phase 2 trial described in Example 3, 22% of subjectswho had a history of gout (20 out of 90 enrolled patients) reported goutflare in the first month (Table 2).

TABLE 1 Flares in subjects with history of gout Subject Flare in 1^(st)month Dose of SEL-037 1 Yes 0.2 mg/kg 2 No 0.2 mg/kg 3 No 0.4 mg/kg 4 No0.2 mg/kg 5 No 0.4 mg/kg 6 No 0.2 mg/kg 7 No 0.4 mg/kg 8 Yes 0.4 mg/kg 9No 0.2 mg/kg 10 No 0.4 mg/kg 11 No 0.2 mg/kg 12 No 0.4 mg/kg 13 No 0.4mg/kg 14 No 0.4 mg/kg 15 No 0.2 mg/kg 16 No 0.2 mg/kg 17 No 0.4 mg/kg 18Yes 0.2 mg/kg 19 No 0.2 mg/kg 20 No 0.4 mg/kg 21 No 0.4 mg/kg 22 Yes 0.4mg/kg 23 No 0.4 mg/kg 24 No 0.2 mg/kg 25 No 0.2 mg/kg 26 Yes 0.4 mg/kg27 No 0.2 mg/kg 28 No 0.2 mg/kg 29 Yes 0.2 mg/kg 30 No 0.2 mg/kg 31 No0.4 mg/kg 32 Yes 0.2 mg/kg 33 Yes 0.2 mg/kg 34 Yes 0.4 mg/kg 35 Yes 0.2mg/kg 36 No 0.2 mg/kg 37 No 0.2 mg/kg 38 No 0.2 mg/kg 39 No 0.4 mg/kg 40No 0.4 mg/kg 41 Yes 0.2 mg/kg 42 No 0.4 mg/kg 43 Yes 0.4 mg/kg 44 No 0.4mg/kg 45 Yes 0.4 mg/kg 46 Yes 0.4 mg/kg 47 No 0.4 mg/kg 48 No 0.4 mg/kg49 Yes 0.4 mg/kg 50 No 0.4 mg/kg 51 No 0.2 mg/kg 52 Yes 0.2 mg/kg 53 No0.2 mg/kg 54 No 0.4 mg/kg 55 No 0.4 mg/kg 56 No 0.4 mg/kg 57 Yes 0.4mg/kg 58 No 0.4 mg/kg 59 No 0.4 mg/kg 60 No 0.4 mg/kg 61 No 0.4 mg/kg 62No 0.4 mg/kg 63 No 0.4 mg/kg 64 No 0.4 mg/kg 65 No 0.2 mg/kg 66 No 0.2mg/kg 67 No 0.4 mg/kg 68 No 0.2 mg/kg 69 Yes 0.4 mg/kg 70 No 0.4 mg/kg71 No 0.4 mg/kg 72 No 0.4 mg/kg 73 Yes 0.4 mg/kg 74 No 0.4 mg/kg 75 No0.4 mg/kg 76 No 0.2 mg/kg 77 No 0.4 mg/kg 78 No 0.4 mg/kg 79 Yes 0.2mg/kg 80 No 0.4 mg/kg 81 No 0.4 mg/kg 82 No 0.4 mg/kg 83 Yes 0.4 mg/kg84 No 0.4 mg/kg 85 No 0.4 mg/kg 86 No 0.4 mg/kg 87 No 0.2 mg/kg 88 No0.2 mg/kg 89 No 0.4 mg/kg 90 No 0.4 mg/kg 91 Yes 0.4 mg/kg 92 No 0.2mg/kg 93 No 0.2 mg/kg 94 No 0.2 mg/kg 95 Yes 0.2 mg/kg 96 No 0.4 mg/kg

TABLE 2 Flares in SEL-212 subjects SEL-212 subjects with gout Flare in1^(st) month Cohort/dose 1 No Cohort 3/SEL-110 0.05 mg/kg; SEL-037 0.2mg/kg 2 No Cohort 4/SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg 3 No Cohort3/SEL-110 0.05 mg/kg; SEL-037 0.2 mg/kg 4 No Cohort 4/SEL-110 0.05mg/kg; SEL-037 0.4 mg/kg 5 No Cohort 5/SEL-110 0.08 mg/kg; SEL-037 0.2mg/kg 6 No Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 7 Yes Cohort10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 8 No Cohort 3/SEL-110 0.05mg/kg; SEL-037 0.2 mg/kg 9 No Cohort 4/SEL-110 0.05 mg/kg; SEL-037 0.4mg/kg 10 No Cohort 7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 11 No Cohort8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg 12 No Cohort 8/SEL-110 0.1 mg/kg;SEL-037 0.4 mg/kg 13 No Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg14 No Cohort 11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 15 No Cohort11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 16 No Cohort 4/SEL-110 0.05mg/kg; SEL-037 0.4 mg/kg 17 Yes Cohort 5/SEL-110 0.08 mg/kg; SEL-037 0.2mg/kg 18 No Cohort 3/SEL-110 0.05 mg/kg; SEL-037 0.2 mg/kg 19 No Cohort4/SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg 20 No Cohort 6/SEL-110 0.08mg/kg; SEL-037 0.4 mg/kg 21 Yes Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4mg/kg 22 No Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg 23 No Cohort7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 24 No Cohort 7/SEL-110 0.1 mg/kg;SEL-037 0.2 mg/kg 25 Yes Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg26 No Cohort 7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 27 No Cohort7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 28 Yes Cohort 7/SEL-110 0.1mg/kg; SEL-037 0.2 mg/kg 29 No Cohort 3/SEL-110 0.05 mg/kg; SEL-037 0.2mg/kg 30 Yes Cohort 3/SEL-110 0.05 mg/kg; SEL-037 0.2 mg/kg 31 YesCohort 4/SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg 32 No Cohort 3/SEL-1100.05 mg/kg; SEL-037 0.2 mg/kg 33 No Cohort 3/SEL-110 0.05 mg/kg; SEL-0370.2 mg/kg 34 No Cohort 5/SEL-110 0.08 mg/kg; SEL-037 0.2 mg/kg 35 NoCohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 36 No Cohort 6/SEL-1100.08 mg/kg; SEL-037 0.4 mg/kg 37 Yes Cohort 5/SEL-110 0.08 mg/kg;SEL-037 0.2 mg/kg 38 No Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg 39Yes Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg 40 No Cohort10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 41 Yes Cohort 10/SEL-110 0.125mg/kg; SEL-037 0.4 mg/kg 42 Yes Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4mg/kg 43 No Cohort 8/SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg 44 No Cohort12/SEL-110 0.15 mg/kg; SEL-037 0.4 mg/kg 45 Yes Cohort 12/SEL-110 0.15mg/kg; SEL-037 0.4 mg/kg 46 No Cohort 12/SEL-110 0.15 mg/kg; SEL-037 0.4mg/kg 47 No Cohort 11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 48 YesCohort 11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 49 No Cohort 7/SEL-1100.1 mg/kg; SEL-037 0.2 mg/kg 50 No Cohort 6/SEL-110 0.08 mg/kg; SEL-0370.4 mg/kg 51 No Cohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 52 YesCohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 53 No Cohort 12/SEL-1100.15 mg/kg; SEL-037 0.4 mg/kg 54 No Cohort 12/SEL-110 0.15 mg/kg;SEL-037 0.4 mg/kg 55 No Cohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg56 No Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 57 No Cohort4/SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg 58 No Cohort 4/SEL-110 0.05mg/kg; SEL-037 0.4 mg/kg 59 No Cohort 5/SEL-110 0.08 mg/kg; SEL-037 0.2mg/kg 60 No Cohort 5/SEL-110 0.08 mg/kg; SEL-037 0.2 mg/kg 61 No Cohort4/SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg 62 No Cohort 3/SEL-110 0.05mg/kg; SEL-037 0.2 mg/kg 63 Yes Cohort 4/SEL-110 0.05 mg/kg; SEL-037 0.4mg/kg 64 No Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 65 No Cohort6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 66 No Cohort 12/SEL-110 0.15mg/kg; SEL-037 0.4 mg/kg 67 Yes Cohort 12/SEL-110 0.15 mg/kg; SEL-0370.4 mg/kg 68 No Cohort 12/SEL-110 0.15 mg/kg; SEL-037 0.4 mg/kg 69 NoCohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 70 No Cohort 11/SEL-1100.15 mg/kg; SEL-037 0.2 mg/kg 71 No Cohort 8/SEL-110 0.1 mg/kg; SEL-0370.4 mg/kg 72 No Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 73 YesCohort 7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 74 No Cohort 8/SEL-110 0.1mg/kg; SEL-037 0.4 mg/kg 75 No Cohort 10/SEL-110 0.125 mg/kg; SEL-0370.4 mg/kg 76 No Cohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 77 YesCohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg 78 No Cohort 12/SEL-1100.15 mg/kg; SEL-037 0.4 mg/kg 79 No Cohort 12/SEL-110 0.15 mg/kg;SEL-037 0.4 mg/kg 80 No Cohort 12/SEL-110 0.15 mg/kg; SEL-037 0.4 mg/kg81 No Cohort 11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 82 No Cohort11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 83 No Cohort 10/SEL-110 0.125mg/kg; SEL-037 0.4 mg/kg 84 No Cohort 10/SEL-110 0.125 mg/kg; SEL-0370.4 mg/kg 85 Yes Cohort 6/SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg 86 NoCohort 7/SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg 87 No Cohort 7/SEL-110 0.1mg/kg; SEL-037 0.2 mg/kg 88 No Cohort 7/SEL-110 0.1 mg/kg; SEL-037 0.2mg/kg 89 Yes Cohort 11/SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg 90 NoCohort 10/SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg

A phase 2 study was undertaken (Example 2). This study involved theadministration of multiple IV infusions of PLA/PLA-PEG syntheticnanocarriers comprising rapamycin together with pegsiticase in order toassess its safety and tolerability. 96 subjects were randomized anddosed, with 27 subjects (28%) reported as receiving gout flareprophylaxis with colchicine/NSAIDs (Table 3).

TABLE 3 Subjects who suffered from gout flare following treatmentSEL-212 subject Cohort Dose Gout flare prophylaxis with colchicine/NSAID1 1 SEL-037 0.2 mg/kg Yes 2 10 SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kgYes 3 5 SEL-110 0.08 mg/kg; SEL-037 0.2 mg/kg Yes 4 8 SEL-110 0.1 mg/kg;SEL-037 0.4 mg/kg Yes 5 8 SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg Yes 6 8SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg Yes 7 7 SEL-110 0.1 mg/kg; SEL-0370.2 mg/kg Yes 8 7 SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg Yes 9 7 SEL-1100.1 mg/kg; SEL-037 0.2 mg/kg Yes 10 3 SEL-110 0.05 mg/kg; SEL-037 0.2mg/kg Yes 11 3 SEL-110 0.05 mg/kg; SEL-037 0.2 mg/kg Yes 12 1 SEL-0370.2 mg/kg Yes 13 4 SEL-110 0.05 mg/kg; SEL-037 0.4 mg/kg Yes 14 1SEL-037 0.2 mg/kg Yes 15 6 SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg Yes 165 SEL-110 0.08 mg/kg; SEL-037 0.2 mg/kg Yes 17 8 SEL-110 0.1 mg/kg;SEL-037 0.4 mg/kg Yes 18 10 SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg Yes19 8 SEL-110 0.1 mg/kg; SEL-037 0.4 mg/kg Yes 20 12 SEL-110 0.15 mg/kg;SEL-037 0.4 mg/kg Yes 21 11 SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg Yes 2210 SEL-110 0.125 mg/kg; SEL-037 0.4 mg/kg Yes 23 4 SEL-110 0.05 mg/kg;SEL-037 0.4 mg/kg Yes 24 12 SEL-110 0.15 mg/kg; SEL-037 0.4 mg/kg No 257 SEL-110 0.1 mg/kg; SEL-037 0.2 mg/kg Yes 26 10 SEL-110 0.125 mg/kg;SEL-037 0.4 mg/kg Yes 27 6 SEL-110 0.08 mg/kg; SEL-037 0.4 mg/kg Yes 2811 SEL-110 0.15 mg/kg; SEL-037 0.2 mg/kg Yes

Flare rates in the above subjects were compared to the flare rates inthe pegloticase trials. Those subjects who received gout flareprophylaxis (with colchicine or NSAIDS) only were chosen to match thepegloticase subject conditions. Flare frequency (number of flares perpatient month) was selected as a measure by which to compare flarerates. This measure was chosen based on the fact that the trial datacovers 5 treatment cycles; while the pegloticase trials varied in lengthfrom 35 days (Sundy et al., Pharmacokinetics and pharmacodynamics ofintravenous PEGylated recombinant mammalian urate oxidase in patientswith refractory gout. Arthritis and Rheumatism. Vol. 56, No. 3, March2007, pp 1021-1028) to 6 months (John S. Sundy, MD, PhD; Herbert S. B.Baraf, MD; Robert A. Yood, MD; et al. Efficacy and Tolerability ofPegloticase for the Treatment of Chronic Gout in Patients Refractory toConventional Treatment: Two Randomized Controlled Trials. JAMA.2011;306(7):711-720). Patient monthly rates were chosen to be able tocompare between trials.

For the SEL-212 study, all subjects who met all inclusion and exclusioncriteria were given pre-medication to prevent gout flare. Thepre-medication regimen began one week prior to the first dosing of studydrug and continued for the duration of the clinical study. Subjects weregiven colchicine (1.2 mg) as a single loading dose. They then continuedto receive colchicine (one 0.6 mg dose daily) for the remainder of theirparticipation in the trial. If there was a contraindication tocolchicine, the subject received ibuprofen (600 mg TID) or an equivalentNSAID unless the subject had a contraindication to NSAIDs generally. Ifthe subject has a contraindication to colchicine and to NSAIDs, then nopre-medication was given. The gout flare prevention medication continuedfor the duration of the clinical study. Subjects who began receiving aNSAID as gout flare prevention medication due to a contraindication tocolchicine or under a previous version of this protocol continued toreceive the NSAID for the duration of the study.

Subjects also received pre-medication with antihistamines and steroids,consisting of 180 mg fexofenadine (oral) twice times (evening before(-12 h ± 2 h, self-administered) and -2 h ± 1 hours before receiving thestudy drug) and 40 mg methylprednisolone (or equivalent drug, forexample prednisone 50 mg IV or dexamethasone 8 mg IV) intravenously -1 h± 0.5 hours before receiving the study drug (i.e., prior to SEL-037 forCohorts 1 and 2 and Part B of all cohorts or prior to SEL-110 forCohorts 3-8, 10, 11, and 12) to reduce infusion reactions.

Cohorts 3 and 4 were grouped together for this analysis, as they weregiven the same dose of synthetic nanocarriers comprising rapamycin (0.05mg/kg), and likewise cohorts 5 and 6 have been grouped together (with asynthetic nanocarrier comprising rapamycin dose of 0.08 mg/kg). Incohorts 1-12, ninety-six subjects have been dosed with a total of 238treatment cycles. Not all subjects received all treatments, as certainsubjects were discontinued following protocol changes. Twenty-ninesubjects had 49 gout flares reported during the 238 treatment cycles.This can be equated to 21 flares per treatment cycle; in other words, aflare frequency of 0.21 flares per patient month.

For comparison, the Phase 3 pegloticase trials (John S. Sundy, MD, PhD;Herbert S. B. Baraf, MD; Robert A. Yood, MD; et al. Efficacy andTolerability of Pegloticase for the Treatment of Chronic Gout inPatients Refractory to Conventional Treatment: Two Randomized ControlledTrials. JAMA. 2011;306(7):711-720) were examined to determine the numberof flares per month and the area under the curve (AUC) of mean serumuric acid (sUA) levels over time (up to 20 weeks). Subjects were given0.6 mg of colchicine once or twice daily (or a nonsteroidalanti-inflammatory drug) one week before first infusion, which continuedthroughout the study. The study also included prophylaxis againstinfusion related reactions (IRs) before each infusion. The subjects wereadministered oral fexofenadine (60 mg) the evening before and againimmediately before, the infusion, as well as acetaminophen 1000 mg) andIV hydrocortisone (200 mg) immediately before the infusion. Data pointswere obtained from Table 2, which presents the biweekly/monthly flareper patient month. Data points were also identified using a graphdigitizer for FIG. 2 of the aforementioned reference. The data wasextrapolated to 20 weeks. The AUC of mean sUA levels over time (up to 20weeks) and the flares per patient month were calculated using thecombined weighted average for responders and non-responders for sUA AUC.The AUC of the biweekly group was 12.3 of responders; 102.0 was the AUCof the biweekly group of non-responders cut to 20 weeks. Similarcalculations were performed to obtain the monthly data.

Further comparisons can be made with the primary branded oral uric acidlowering medication, febuxostat. In a phase 3, randomized, double-blind,multi-center trial, the safety and efficacy of febuxostat was studiedover 52 weeks (Michael A. Becker, M.D., H. Ralph Schumacher, Jr., M.D.,Robert L. Wortmann, M.D., Patricia A. MacDonald, B.S.N., N.P., DeniseEustace, B.A., William A. Palo, M.S., Janet Streit, M.S., and NancyJoseph-Ridge, M.D. Febuxostat Compared with Allopurinol in Patients withHyperuricemia and Gout. N Engl J Med 2005; 353:2450-2461Dec. 8, 2005).The comparison period for this analysis included only the first 8 weeksof that study, when gout flare prophylaxis (naproxen or colchicine) wasadministered. Data points were identified using a graph digitizer forFIG. 1 of the above reference. At a dose of 80 mg/day, 55 out of 255subjects required treatment for at least one gout flare. This would bethe equivalent to a flare frequency of at least 0.22 flares per patientmonth, and possibly more. At a dose of 120 mg/day, 90 out of 250subjects required treatment for at least one gout flare, equating to atleast a flare frequency of 0.36 flares per patient month, and possiblymore.

The tabulated data outlining the comparison in flare frequency betweenthe different medications alongside their efficacy in reducing serumuric acid (sUA) is compiled in Table 4.

TABLE 4 Flares per patient month compared with other uric acid loweringtreatments Medication and dosage Flares per patient month Area UnderCurve of Mean sUA Levels Over Time Up to 20 Weeks Flares per patientmonth * AUC SEL-212 monthly 0.21 15.3 3.21 Pegloticase biweekly 0.5264.0 33.28 Pegloticase monthly 0.70 94.4 66.05 Febuxostat 80 mg/day 0.22108.3 23.83 Febuxostat 120 mg/day 0.36 93.4 33.62

The flare frequency is clearly reduced for the subjects who received therapamycincontaining nanocarrier concomitantly administered withpegsiticase as compared to all of the other medications. This unexpectedoutcome is significantly better than with other therapies as shown inTable 4. This also has the benefit for patient adherence to uric acidlowering therapies, such as uricase, as adherence is greatly reducedwhen rebound flares occur following initiation of therapy (Treatment ofchronic gouty arthritis: it is not just about uratelowering therapy.Schlesinger N - Semin. Arthritis Rheum. - Oct. 1, 2012; 42 (2); 155-65).In addition, a combination assessment of glare frequency as well as thearea under the cruve of mean sUA levels over time show both improvedefficacy and reduced gout flares. This finding was surprising, astypically improved efficacy in sUA removal can result in increased goutflares. Here, with the SEL-212 study drug and anti-inflammatorytreatment, both efficacy and gout flare reduction can be achieved.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A method, comprising: concomitantly administering to a subject inneed thereof 1) a composition comprising synthetic nanocarrierscomprising an immunosuppressant and 2) a composition comprising anuricase; and further comprising administering 3) a compositioncomprising an anti-inflammatory therapeutic, wherein the compositioncomprising an anti-inflammatory therapeutic is administeredconcomitantly with the composition comprising synthetic nanocarrierscomprising an immunosuppressant and the composition comprising anuricase.
 2. The method of claim 1, wherein the composition comprising ananti-inflammatory therapeutic is administered prior to the compositioncomprising synthetic nanocarriers comprising an immunosuppressant andthe composition comprising an uricase.
 3. The method of claim 1, whereinthe anti-inflammatory therapeutic is administered at least once prior.4. The method of claim 1, wherein the anti-inflammatory therapeutic isadministered a week prior.
 5. The method of claim 1, wherein theanti-inflammatory therapeutic is a NSAID.
 6. The method of claim 1,wherein the anti-inflammatory therapeutic is colchicine or ibuprofen. 7.The method of claim 1, wherein the method further comprisesadministering to the subject one or more compositions comprising aninfusion reaction therapeutic.
 8. The method of claim 7, wherein the oneor more compositions comprising an infusion reaction therapeuticcomprises an antihistamine and/or a corticosteroid.
 9. The method ofclaim 8, wherein the antihistamine is fexofenadine.
 10. The method ofclaim 8, wherein the corticosteroid is methylprednisolone, prednisone ordexamethasone.
 11. The method of claim 7, wherein the one or morecompositions comprising an infusion reaction therapeutic is/areadministered at least once prior to the composition comprising syntheticnanocarriers comprising an immunosuppressant and the compositioncomprising an uricase.
 12. The method of claim 11, wherein thecomposition(s) comprising an infusion reaction therapeutic isadministered at least twice prior to the composition comprisingsynthetic nanocarriers comprising an immunosuppressant and thecomposition comprising an uricase.
 13. The method of claim 7, whereinthe composition(s) comprising an infusion reaction therapeutic isadministered within 24 hours of the composition comprising syntheticnanocarriers comprising an immunosuppressant and the compositioncomprising an uricase.
 14. The method of claim 1, wherein the uricase ispegylated uricase.
 15. The method of claim 14, wherein the pegylateduricase is pegadricase or pegloticase.
 16. The method of claim 1,wherein the immunosuppressant is an mTOR inhibitor.
 17. The method ofclaim 16, wherein the mTOR inhibitor is a rapalog.
 18. The method ofclaim 17, wherein the rapalog is rapamycin.
 19. The method of claim 1,wherein the subject is human. 20-49. (canceled)
 50. A composition,comprising: 1) a composition comprising synthetic nanocarrierscomprising an immunosuppressant and 2) a composition comprising anuricase; and further comprising administering 3) a compositioncomprising an anti-inflammatory therapeutic. 51-83. (canceled)